Isolated GRP94 ligand binding domain polypeptide and nucleic acid encoding same, crystalline form of same, and screening methods employing same

ABSTRACT

An isolated GRP94 ligand binding domain polypeptide, a three-dimensional crystal structure of the same, and methods of using the same to design modulators of Hsp90 proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to U.S. ProvisionalPatent Application Ser. No. 60/326,291, filed Oct. 1, 2001 and entitled“ISOLATED GRP94 LIGAND BINDING DOMAIN POLYPEPTIDE AND NUCLEIC ACIDENCODING SAME, CRYSTALLINE FORM OF SAME, AND SCREENING METHODS EMPLOYINGSAME”, herein incorporated by referenced in its entirety.

TECHNICAL FIELD

The present invention relates to compositions and methods pertaining tothe modulation of molecular chaperone function by regulatory ligands. Ina preferred embodiment, the present invention relates to an isolated andpurified GRP94 ligand binding domain (LBD) polypeptide, a crystallizedform of the GRP94 LBD in complex with a ligand, and to screening methodsassociated therewith.

Table of Abbreviations 8-ANS 1,8-anilinonaphthalenesulfonate APC antigenpresenting cells BiP ER hsp70 homolog bis-ANS4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid BMDC bonemarrow-derived dendritic cells BN-PAGE blue native polyacrylamide gelelectrophoresis CEA carcinoembryonic antigen(s) CT computed tomographicCTL cytotoxic T lymphocyte(s) DC dendritic cells DMEM Dulbecco'smodified Eagle's medium DTH delayed-type hypersensitivity ER endoplasmicreticulum GALT gut-associated lymphoid tissue GRP94 glucose regulatedprotein of 94 kDa, ER paralog of the Hsp90 family of chaperones GSTglutathione S-transferase HIV human immunodeficiency virus HPLC highpressure liquid chromatography hr hour(s) hsp(s) heat shock protein(s)HSP70 heat shock protein of 70 kDa Hsp90 any member of the Hsp90 familyof chaperones HSP90 heat shock protein of 90 kDa HSV herpes simplexvirus IFN interferon Ig immunoglobulin IGF-1 insulin-like growth factorIgG immunoglobulin G IL interleukin LBD ligand binding domain MHC majorhistocompatability complex min minute MLTC mixed lymphocyte tumor cellassay NECA N-ethylcarboxamidoadenosine PDI protein disulfide isomerasePSA prostate-specific antigen RSV respiratory syncytial virus RT roomtemperature SDS-PAGE sodium dodecyl sulfate-polyacrylamide gelelectrophoresis TAP transporter associated with antigen presentationcomplex TFA trifluoroacetic acid TNF tumor necrosis factor

Amino Acid Abbreviations Single-Letter Code Three-Letter Code Name A AlaAlanine V Val Valine L Leu Leucine I Ile Isoleucine P Pro Proline F PhePhenylalanine W Trp Tryptophan M Met Methionine G Gly Glycine S SerSerine T Thr Threonine C Cys Cysteine Y Tyr Tyrosine N Asn Asparagine QGln Glutamine D Asp Aspartic Acid E Glu Glutamic Acid K Lys Lysine R ArgArginine H His Histidine

Functionally Equivalent Codons Amino Acid Codons Alanine Ala A GCA GCCGCG GCU Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAU Glumatic acidGlu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCCCCG CCU Glutamine Gln Q CAA CAG Threonine Thr T ACA ACC ACG ACU ValineVal V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAULeucine Leu L UUA UUG CUA CUC CUG CUU Arginine Arg R AGA AGG CGA CGC CGGCGU Serine Ser S ACG AGU UCA UCC UCG UCU

BACKGROUND ART

The pursuit of approaches for treatment and prevention of cancer andinfectious diseases represents an ongoing effort in the medicalcommunity. Recent efforts to combat cancer and infectious disease haveincluded attempts to induce or enhance immune responses in subjectssuffering from a type of cancer or an infectious disease. See, e.g.Srivastava et al. (1998) Immunity 8:657-665.

Ischemia/reperfusion injury is a significant source of morbidity andmortality in a number of clinical disorders, including myocardialinfarction, cerebrovascular disease, and peripheral vascular disease. Inaddition, ischemia/reperfusion is relevant to the function oftransplanted organs and to the recovery expedience following anycardiovascular surgery. See Fan et al. (1999) J Mol Med 77:577-596.Thus, the identification of cellular protective mechanisms againstischemia-induced damage is a central goal for therapy of, for example,heart attacks, strokes, and neurodegenerative diseases, as well as forimprovement of recovery following surgery or transplantation.

The Hsp90 class of molecular chaperones are among the most abundantproteins in eukaryotic cells. Hsp90 family members are phylogeneticallyubiquitous whereas the endoplasmic reticulum paralog of HSP90, GRP94(gp96, ERp99, endoplasmin), is found only in higher plants and metazoans(Nicchitta (1998) Curr Opin Immunol 10:103-109). The Hsp90 family ofproteins are known to be involved in directing the proper folding andtrafficking of newly synthesized proteins and in conferring protectionto the cell during conditions of heat shock, oxidative stress, nutrientstress, and other physiological stress scenarios (Toft (1998) TrendsEndocrinol Metab 9:238-243; Pratt (1998) Proc Soc Exp Biol Med217:420-434). Under such stress conditions, protein folding, proteinoligomeric assembly, and protein stability can be profoundly disrupted.It is the function of the Hsp90 family of proteins, in concert withother molecular chaperones, to assist in preventing and reversingstress-induced inactivation of protein structure and function.

At a molecular level, HSP90 function in protein folding is known torequire the activity of a series of co-chaperones and accessorymolecules, including Hsp70, p48Hip, p60Hop, p23, and FKBP52 (Prodromouet al. (1999) EMBO J 18:754-762; Johnson et al. (1996) J Steroid BiochemMol Biol 56:31-37; Chang et al. (1997) Mol Cell Biol 17:318-325; Duinaet al. (1996) Science 274:1713-1715; Chen et al. (1996) Mol Endocrinol10:682-693; Smith et al. (1993) J Biol Chem 268:18365-18371; Dittmar etal. (1998) J Biol Chem 273:7358-7366; Kosano et al. (1998) J Biol Chem273:3273-3279). These co-chaperones and accessory molecules participatein both concerted and sequential interactions with HSP90 and therebyserve to regulate its chaperone activity (Buchner (1999) Trends BiochemSci 24:136-141; Pratt et al. (1996) Exs 77:79-95; Pratt (1998) Proc SocExp Biol Med 217:420-434; Caplan (1999) Trends Cell Biol 9:262-268).

In addition to the contribution of co-chaperone proteins to theregulation of HSP90 function, recent crystallographic studies haveidentified an ATP/ADP binding pocket in the N-terminal domain of yeastand human HSP90, suggesting that HSP90 activity is regulated throughcyclic ATP binding and hydrolysis, as has been established for the Hsp70family of chaperones (Kassenbrock & Kelly (1989) EMBO J. 8:1461-1467;Flynn et al. (1989) Science 245:385-390; Palleros et al. (1991) ProcNatl Acad Sci USA 88:519-523; Sriram et al. (1997) Structure 5:403-14;Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J CellBiol 143:901-910; Csermely & Kahn (1991) J Biol Chem 266:4943-4950;Csermely et al. (1993) J Biol Chem 268:1901-1907; Sullivan et al. (1997)J Biol Chem 272:8007-8012; Scheibel et al. (1997) J Biol Chem272:18608-18613; Scheibel et al. (1998) Proc Natl Acad Sci USA95:1495-1499; Panaretou et al. (1998) EMBO J 17:4829-4836; Caplan (1999)Trends Cell Biol 9:262-268; Grenert et al. (1999) J Biol Chem274:17525-17533).

It has also been reported that HSP90 contains motifs bearing significantsimilarities to the Walker “A” and “B” sequences associated with ATPbinding (Csermely & Kahn (1991) J Biol Chem 266:4943-4950; Jakob et al.(1996) J Biol Chem 271:10035-10041). Although these sequences aresubstantially different from the consensus sequences found among serineand tyrosine kinases, they are homologous to the ATP binding sequenceseen in the Hsp70 family of proteins (Csermely & Kahn (1991) J Biol Chem266:4943-4950). Consistent with sequence predictions, ATP binding,autophosphorylation activity, and ATPase activity have all beendemonstrated for HSP90, though these findings are not withoutcontroversy (Csermely & Kahn (1991) J Biol Chem 266:4943-4950; Nadeau etal. (1993) J Biol Chem 268:1479-1487, Jakob et al. (1996) J Biol Chem271:10035-10041; Grenert et al. (1999) J Biol Chem 274:17525-17533;Scheibel et al. (1997) J Biol Chem 272:18608-18613; Prodromou et al.(1997) Cell 90:65-75).

In part because of the very low affinity of HSP90 for ATP, a role forATP in the regulation of HSP90 function remained under question untilcrystallographic resolution of the N-terminal domain of yeast and humanHSP90 in association with bound adenosine nucleotides (Prodromou et al.(1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910).Aided by atomic scale structural insights, amino acid residues criticalfor ATP binding and hydrolysis were subsequently identified and analyzed(Prodromou et al. (1997) Cell 90:65-75; Panaretou et al. (1998) EMBO J17:4829-4836; Obermann et al. (1998) J Cell Biol 143:901-910). Thus, inthe human HSP90, aspartate 93 (D128 for GRP94; D79 for yeast HSP90)provides a direct hydrogen bond interaction with the N6 group of thepurine moiety of the adenosine ring and is essential for ATP binding(Prodromou et al. (1997) Cell 90:65-75; Obermann et al. (1998) J CellBiol 143:901-910). Glutamate 47 (E82 for GRP94; E33 for yeast HSP90) waspostulated to play an important catalytic role in ATP hydrolysis, basedboth on its location relative to bound nucleotide and through comparisonwith the ATP binding domain of E. coli DNA gyrase B (Prodromou et al.(1997) Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910).In subsequent mutagenesis studies of yeast HSP90, it was observed thatthe D79 mutant was deficient in ATP binding and that E47 mutants weredeficient in ATP hydrolysis activity (Obermann et al. (1998) J Cell Biol143:901-910; Panaretou et al. (1998) EMBO J 17:4829-4836). As furtherevidence for a function of these residues in HSP90 activity, yeastcontaining either mutant form of HSP90 were inviable (Obermann et al.(1998) J Cell Biol 143:901-910; Panaretou et al. (1998) EMBO J17:4829-4836).

Progress in the development of Hsp90-based therapeutic and otherapplications has been impeded by a lack of characterization of ligandinteractions of Hsp90 proteins, including GRP94. Despite theabove-described characterization of ATP interaction with HSP90, evidencein support of intrinsic ATP binding and ATPase activities with respectto GRP94 is controversial and, as with HSP90, a clear consensusregarding the molecular basis of an adenosine nucleotide-mediatedregulation of GRP94-substrate interactions has yet to emerge (Jakob etal. (1996) J Biol Chem 271:10035-10041; Wearsch & Nicchitta (1997) JBiol Chem 272:5152-5156; Li and Srivastava (1993) EMBO J 12:3143-3151;Csermely et al. (1995) J Biol Chem 270:6381-6388; Csermely et al. (1998)Pharmacol Ther 79:129-168).

What is needed, then, is characterization of ligand interactions at theligand binding pocket of a HSP90 protein, in particular GRP94 and HSP90.To this end, the present invention discloses an isolated and purifiedGRP94 LBD polypeptide as well as X-ray crystallographic methods thathave been used to elucidate GRP94 LBD three-dimensional structure. Priorto the disclosure of the present application, high-quality GRP94crystals as required for X-ray crystallographic experiments have beenunavailable. The solved crystal structure of GRP94 provides structuraldetails that facilitate the design of Hsp90 protein modulators. Usingsuch methods, the active and inactive structural conformations of GRP94and HSP90 are elucidated, and the regulative capacity of severalcompounds to induce the active or inactive conformation is alsodemonstrated. The disclosure herein also provides drug screening methodspertaining to the biological activity of Hsp90 proteins. Thus, thepresent invention meets a long-standing need in the art for methods andcompositions that contribute to the understanding, diagnosis andtreatment of disorders related to Hsp90 protein function.

SUMMARY OF THE INVENTION

A substantially pure GRP94 ligand binding domain polypeptide incrystalline form is disclosed. In one embodiment, the crystalline formhas unit cell has lattice constants of a=99.889 Å, b=89.614 Å, c=60.066Å; β=90.132°; space group symmetry C2; and 2 GRP94+NECA complexes in theasymmetric unit. In another embodiment, the crystalline form has latticeconstants of the unit cell has lattice constants of a=89.200 Å, b=99.180Å, c=63.071 Å; α=β=γ=90.0°; space group symmetry C222₍₁₎; and 1GRP94+NECA complex in the asymmetric unit.

Optionally, the GRP94 polypeptide has the amino acid sequence of SEQ IDNO:6. Preferably, the GRP94 polypeptide is in complex with a ligand,more preferably the ligand is NECA. Most preferably, the GRP94polypeptide has a crystalline structure further characterized by thecoordinates corresponding to Tables 1, 2 or 3.

A method for determining the three-dimensional structure of acrystallized GRP94 ligand binding domain polypeptide to a resolution ofabout 1.8 Å or better is also disclosed. The method comprises: (a)crystallizing a GRP94 ligand binding domain polypeptide; and (b)analyzing the GRP94 ligand binding domain polypeptide to determine thethree-dimensional structure of the crystallized GRP94 ligand bindingdomain polypeptide, whereby the three-dimensional structure of acrystallized GRP94 ligand binding domain polypeptide is determined to aresolution of about 1.8 Å or better.

A method of generating a crystallized GRP94 ligand binding domainpolypeptide is also disclosed. In a preferred embodiment, the methodcomprises: (a) incubating a solution comprising a GRP94 ligand bindingdomain with a reservoir; and (b) crystallizing the GRP94 ligand bindingdomain polypeptide using the hanging drop method, whereby a crystallizedGRP94 ligand binding domain polypeptide is generated.

Further, a method of designing a modulator of an Hsp90 protein isdisclosed. The method comprises: (a) designing a potential modulator ofan Hsp90 protein that will make interactions with amino acids in theligand binding site of the Hsp90 protein based upon the atomic structurecoordinates of a GRP94 ligand binding domain polypeptide; (b)synthesizing the modulator; and (c) determining whether the potentialmodulator modulates the activity of the Hsp90 protein, whereby amodulator of an Hsp90 protein is designed.

Additionally, a method of designing a modulator that selectivelymodulates the activity of a GRP94 polypeptide is disclosed. The methodcomprises: (a) obtaining a crystalline form of a GRP94 ligand bindingdomain polypeptide; determining the three-dimensional structure of thecrystalline form of the GRP94 ligand binding domain polypeptide; and (c)synthesizing a modulator based on the three-dimensional structure of thecrystalline form of the GRP94 ligand binding domain polypeptide, wherebya modulator that selectively modulates the activity of a GRP94polypeptide is designed.

A method for identifying a GRP94 modulator is also disclosed. The methodcomprises: (a) providing atomic coordinates of a GRP94 ligand bindingdomain to a computerized modeling system; and (b) modeling ligands thatfit spatially into the binding pocket of the GRP94 ligand binding domainto thereby identify a GRP94 modulator.

A method of identifying a modulator that selectively modulates theactivity of a GRP94 polypeptide compared to other Hsp90 polypeptides isalso disclosed. The method comprises: (a) providing atomic coordinatesof a GRP94 ligand binding domain polypeptide to a computerized modelingsystem; and (b) modeling a ligand that fits into the binding pocket of aGRP94 ligand binding domain and that interacts with conformationallyconstrained residues of a GRP94 ligand binding domain polypeptideconserved among Hsp90 subtypes, whereby a modulator that selectivelymodulates the activity of a GRP94 polypeptide compared to other Hsp90polypeptides is identified.

Further, a method of identifying an Hsp90 modulator that selectivelymodulates the biological activity of one Hsp90 polypeptide compared toGRP94 is disclosed. The method comprises: (a) providing an atomicstructure coordinate set describing a GRP94 ligand binding domainpolypeptide and at least one other atomic structure coordinate setdescribing an Hsp90 ligand binding domain, each ligand binding domaincomprising a ligand binding site; (b) comparing the atomic structurecoordinate sets to identify at least one difference between the sets;(c) designing a candidate ligand predicted to interact with thedifference of step (b); (d) synthesizing the candidate ligand; and (e)testing the synthesized candidate ligand for an ability to selectivelymodulate an Hsp90 as compared to GRP94, whereby an Hsp90 modulator thatselectively modulates the biological activity of the Hsp90 compared toGRP94 is identified.

Additionally, a method of designing a modulator of a GRP94 polypeptideis disclosed. The method comprises: (a) selecting a candidate GRP94ligand; (b) determining which amino acid or amino acids of a GRP94polypeptide interact with the ligand using a three-dimensional model ofa crystallized protein comprising a GRP94 ligand binding domainpolypeptide; (c) identifying in a biological assay for GRP94 activity adegree to which the ligand modulates the activity of the GRP94polypeptide; (d) selecting a chemical modification of the ligand whereinthe interaction between the amino acids of the GRP94 polypeptide and theligand is predicted to be modulated by the chemical modification; (e)synthesizing a chemical compound with the selected chemical modificationto form a modified ligand; (f) contacting the modified ligand with theGRP94 polypeptide; (g) identifying in a biological assay for GRP94activity a degree to which the modified ligand modulates the biologicalactivity of the GRP94 polypeptide; and (h) comparing the biologicalactivity of the GRP94 polypeptide in the presence of modified ligandwith the biological activity of the GRP94 polypeptide in the presence ofthe unmodified ligand, whereby a modulator of a GRP94 polypeptide isdesigned.

An assay method for identifying a compound that inhibits binding of aligand to a GRP94 polypeptide is also disclosed. The assay methodcomprises: (a) designing a test inhibitor compound based on the threedimensional atomic coordinates of a GRP94 ligand binding domainpolypeptide; incubating a GRP94 polypeptide with a ligand in thepresence of a test inhibitor compound; determining an amount of ligandthat is bound to the GRP94 polypeptide, wherein decreased binding ofligand to the GRP94 protein in the presence of the test inhibitorcompound relative to binding of ligand in the absence of the testinhibitor compound is indicative of inhibition; and identifying the testcompound as an inhibitor of ligand binding if decreased ligand bindingis observed, whereby a compound that inhibits binding of a ligand to aGRP94 polypeptide is identified.

A method of screening a plurality of compounds for a modulator of aGRP94 ligand binding domain polypeptide is also provided. The methodcomprises: (a) providing a library of test samples; (b) contacting aGRP94 ligand binding domain polypeptide with each test sample; (c)detecting an interaction between a test sample and the GRP94 ligandbinding domain polypeptide; (d) identifying a test sample that interactswith the GRP94 ligand binding domain polypeptide; and (e) isolating atest sample that interacts with the GRP94 ligand binding domainpolypeptide, whereby a plurality of compounds is screened for amodulator of a GRP94 ligand binding domain polypeptide.

An isolated GRP94 LBD polypeptide is also disclosed. In one embodiment,the isolated polypeptide has the sequence of any of SEQ ID NOs:4 or 6.An isolated nucleic acid molecule encoding a GRP94 LBD polypeptide isalso disclosed, as is a chimeric gene comprising the nucleic acidmolecule to a heterologous promoter, a vector comprising the chimericgene, and a host cell comprising the chimeric gene. Methods of detectingthe GRP LBD polypeptide and nucleic acid encoding the same are alsodisclosed, as is an antibody that specifically recognizes a GRP94 LBDpolypeptide.

A method for identifying a substance that modulates GRP94 LBD functionis also disclosed. The method comprises: (a) isolating a GRP94 LBDpolypeptide; (b) exposing the isolated GRP94 polypeptide to a pluralityof substances; (c) assaying binding of a substance to the isolated GRP94polypeptide; and (d) selecting a substance that demonstrates specificbinding to the isolated GRP94 LBD polypeptide.

Accordingly, it is an object of the present invention to provide a threedimensional structure of the ligand binding domain of a GRP94. This andother objects are achieved in whole or in part by the present invention.

An object of the invention having been stated hereinabove, other objectswill be evident as the description proceeds, when taken in connectionwith the accompanying Drawings and Laboratory Examples as best describedhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph depicting Prodan binding to GRP94 independent ofGRP94 structural state. Fluorescence emission wavelength scans of 0.5 μMnative or heat shocked (hs) GRP94 were performed following exposure to 5μM Prodan for 30 minutes. Values represent the maximal fluorescencerelative to that occurring with an identical concentration of heatshocked GRP94. Experiments were conducted at excitation wavelengths of360 nm (Prodan). All spectra were background corrected.

FIG. 1B is a graph depicting 8-ANS binding to GRP94, and dependence ofsuch binding on GRP94 structural state. Fluorescence emission wavelengthscans of 0.5 μM native or heat shocked (hs) GRP94 were performedfollowing exposure to 5 μM 8-ANS for 30 minutes. Values represent themaximal fluorescence relative to that occurring with an identicalconcentration of heat shocked GRP94. Experiments were conducted atexcitation wavelengths of 372 nm (8-ANS). All spectra were backgroundcorrected.

FIG. 1C is a graph depicting bis-ANS binding to GRP94, and dependence ofsuch binding on GRP94 structural state. Fluorescence emission wavelengthscans of 0.5 μM native or heat shocked (hs) GRP94 were performedfollowing exposure to 5 μM bis-ANS for 20 hours. Values represent themaximal fluorescence relative to that occurring with an identicalconcentration of heat shocked GRP94. Experiments were conducted atexcitation wavelengths of 393 nm (bis-ANS). All spectra were backgroundcorrected.

FIG. 1D is a graph depicting a time course of bis-ANS binding to GRP94.Values represent the maximal fluorescence relative to that occurringwith an identical concentration of heat shocked GRP94. Experiments wereconducted at excitation wavelengths of 393 nm (bis-ANS). All spectrawere background corrected.

FIG. 2A is a graph depicting kinetic analysis of bis-ANS interactionswith heat shocked GRP94. The concentration dependence of bis-ANS bindingto heat shocked GRP94 was conducted under experimental conditions offixed bis-ANS concentration (50 nM) and increasing GRP94 concentration,as indicated.

FIG. 2B is a Klotz plot representation of bis-ANS/GRP94 binding data.Half maximal binding occurs at 110 nM GRP94. Excitation wavelenth, 393nm. Emission wavelength, 475 nm.

FIG. 3 is a digital image of a Coomassie Blue stained gel depicting thatbis-ANS and heat shock increase GRP94 proteolysis sensitivity. GRP94 (5μg, 5 μM) was incubated with 50 μM bis-ANS for one hour at 37° C. orheat shocked for 15 minutes at 50° C. Samples were then digested with0.1% trypsin for 30 minutes at 37° C. and analyzed on 12.5% SDS-PAGEgels. Lane 1, 5 μg of undigested GRP94; lane 2, control native GRP94incubated with trypsin; lane 3, bis-ANS treated GRP94 digested withtrypsin; lane 4, GRP94 heat shocked then digested with trypsin.

FIG. 4 is a digital image of a Coomassie Blue stained gel depicting thatbis-ANS and heat shock induce GRP94 multimerization. GRP94 was heatshocked at 50° C. for 0-15 minutes or incubated with 10-fold molarexcess of bis-ANS and the structural state of the protein analyzed on5-18% native blue polyacrylamide gradient gels. The mobilities of GRP94dimers, tetramers, hexamers, and octamers are shown. Molecular weightstandards are indicated to the right of FIG. 4.

FIG. 5 is a graph depicting that circular dichroism spectra of native,heat shocked, and bis-ANS treated GRP94 are identical. Circulardichroism spectra of 1 μM GRP94 native (diamonds); heat shocked (dot anddash); and treated 2 hours with 10 μM bis-ANS (dotted) are shown.Spectra were collected as described in Examples 1-8 below.

FIG. 6A is a digital image of a Coomassie Blue stained gel depictingthat radicicol blocks bis-ANS structural transitions. GRP94 (5 μM) waspreincubated for one hour at 37° C. with 0-500 μM radicicol andsubsequently incubated for one hour at 37° C. with 50 μM bis-ANS,trypsinized, and the trypsin digestion pattern analyzed by SDS-PAGE.

FIG. 6B is a graph depicting that radicicol blocks heat shock andbis-ANS binding. GRP94 (0.5 μM) was preincubated with 0-10 μM radicicolfor one hour, heat shocked, and subsequently incubated with 1 μMbis-ANS. Bis-ANS binding was determined by spectrofluorometry withbis-ANS binding to native GRP94 in the absence of radicicol shown-forcomparison. Excitation 393 nm, emission 410-600 nm.

FIG. 7A is a graph depicting that bis-ANS and heat shock stimulate GRP94chaperone activity. Citrate synthase enzyme was diluted to 0.15 μM intobuffer containing no GRP94, 1 μM native GRP94, heat shocked GRP94, orGRP94 which had been preincubated for two hours with 10 μM bis-ANS, andcitrate synthase aggregation at 43° C. was monitored by light scatteringat 500 nm in a thermostatted spectrofluorometer.

FIG. 7B is a bar graph depicting that bis-ANS and heat shock stimulateGRP94 peptide binding activity. Native, heat shocked, or bis-ANS treatedGRP94 were incubated with a 10-fold molar excess of ¹²⁵I-VSV8 peptidefor 30 minutes at 37° C. Free peptide was removed by spin columnchromatography and bound radioactive peptide quantitated by gammacounting.

FIG. 8 is a bar graph depicting that GRP94 and Hsp90 exhibitdifferential ligand binding. NECA and ATP binding to GRP94 was performedin the presence of 20 nM [³H]-NECA (closed bars) or 50 μM [³²P]ATP(hatched bars) for 1 hour at 4° C. Bound versus free nucleotide wereseparated by vacuum filtration. PEI treated glass filters (S&S #32,Schleicher and Schuell of Keene, New Hampshire) were used for the NECAbinding assay while nitrocellulose filters (S&S BA85, Schleicher andSchuell of Keene, N.H.) were used to measure ATP binding. The datapresented are averages of triplicate points and are corrected fornonspecific ligand binding.

FIG. 9A is a Scatchard plot depicting characterization of NECA bindingto GRP94. GRP94 was incubated with increasing concentrations of NECA for1 hour at 4° C. as described in Materials and Methods. Bound versus freeNECA were then separated by vacuum filtration with glass filterspretreated in 0.3% PEI.

FIG. 9B is a saturation curve depicting characterization of NECA bindingto GRP94. The curve is plotted with respect to GRP94 dimerconcentration. The maximal binding stoichiometry is 1 molecule of NECAper molecule of GRP94 dimer.

FIG. 9C is a graph depicting stoichiometry of GRP94 binding to NECA(solid oval) and radicicol (solid rectangle). NECA and radicicol bindingto GRP94 was assayed by isothermal titration calorimetry. GRP94 waspresent at a concentration of 5 μM. NECA titrations were performed witha 152 μM NECA stock whereas radicicol titrations were performed with a115 μM stock., ITC data were collected as μcal/sec versus time and thearea under individual injection peaks, determined with the instrumentsoftware, was plotted.

FIG. 10A is a graph depicting a competition assay for NECA by the Hsp90family inhibitors, geldanamycin (♦) and radicicol (▪). GRP94 wasincubated with 20 nM [³H]-NECA and increasing concentrations ofcompetitors for 1 hour at 4° C. Bound NECA was separated from free byvacuum filtration with glass filters pre-treated in 0.3% PEI. All datapoints represent the average of triplicates points minus background(nonspecific NECA binding in the absence of protein).

FIG. 10B is a graph depicting a competition assay for NECA by ATP (♦),ADP (▪), and AMP (▴). GRP94 was incubated with 20 nM 3H-NECA andincreasing concentrations of competitors for 1 hour at 4° C. Bound NECAwas separated from free by vacuum filtration with glass filterspre-treated in 0.3% PEI. All data points represent the average oftriplicate points minus background (nonspecific NECA binding in theabsence of protein).

FIG. 10C is a graph depicting a competition assay for NECA by adenosine(▴), and cAMP (▪). GRP94 was incubated with 20 nM [³H]-NECA andincreasing concentrations of competitors for 1 hour at 4° C. Bound NECAwas separated from free by vacuum filtration with glass filterspre-treated in 0.3% PEI. All data points represent the average oftriplicates points minus background (nonspecific NECA binding in theabsence of protein).

FIG. 11 is a bar graph depicting that ligand binding specificity ofGRP94 to the adenosine base. GRP94 was incubated with 20 nM [³H]-NECAand competitors, all at 50 μM final concentration for 1 hour at 4° C.,and bound vs. free NECA was separated by vacuum filtration with glassfilters pretreated in 0.3% PEI.

FIG. 12 is a graph depicting that binding of ATP, ADP, and AMP to GRP94is sensitive to Mg²⁺ concentration. GRP94 was incubated for 1 hour at 4°C. in 50 mM Tris, 20 nM [³H]-NECA and one of the followingconcentrations of competitor: 3.1×10⁻⁶ M ATP, 3.1×10⁻⁵ M ADP, 6×10⁴ MAMP, or 3.1×10⁻⁵ M adenosine. Reactions were performed in the presenceof 10 mM Mg(OAc)₂ (hatched bars) or in the presence of nominal,endogenous magnesium (closed bars). Bound vs. free NECA was separated byvacuum filtration with glass filters pretreated in 0.3% PEI.

FIG. 13A is a bar graph depicting the effects of NECA on GRP94autophosphorylation. 25 μl reactions consisting of 1 μM GRP94 (closedbars), 0.15 mM γ-³²PATP (6000 cpm/pmol), 10 mM Mg(OAc)₂, and 50 mMK-Hepes, pH 7.4) were incubated for 1 hour at 37° C. One (1) unit caseinkinase II (hatched bars) was incubated in the above conditions with theaddition of 4 μM casein. Competitors were added to the appropriatesamples with a final concentration of 180 μM NECA in 3.6% DMSO, 180 μMradicicol in 3.6% DMSO, 5 μg/ml heparin, 5 mM GTP, or 3.6% DMSO.Phosphorylated species were quantitated on a Fuji MACBAS1000™phosphorimaging system, and the average PSL units of three independentexperiments are displayed.

FIG. 13B is a bar graph depicting ATP hydrolysis in the presence andabsence of GRP94. 100 μl reactions consisting of 1 μM GRP94 monomer,various concentrations of MgATP (pH 7.0), and 50 mM K-Hepes, pH 7.4,were incubated for two hours at 37° C. ATP and ADP were separated on aHewlett Packard HPLC using a Partisil SAX column. Spontaneous ATPhydrolysis was determined in the absence of protein. Hydrolysis in thepresence of GRP94 is indicated by closed bars and spontaneous hydrolysisis indicated by the hatched bars.

FIG. 14 is a graph depicting ligand-induced conformational changes ofGRP94. GRP94 (50 μg/ml) was incubated in buffer A supplemented with 10mM Mg(OAc)₂ and the following concentrations of ligands for 1 hour at37° C.: 50 μM NECA, 50 μM geldanamycin, 2.5 mM ATP, or 2.5 mM ADP.Samples were excited at a wavelength of 295 nm and the tryptophanemission spectra were recorded from 300-400 nm. All spectra werecorrected by subtraction of spectra obtained in buffer alone orbuffer+ligand samples.

BRIEF DESCRIPTION OF SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NOs:1 and 2 are, respectively, a DNA sequence encoding a wildtype full-length human GRP94 (GenBank® Accession No. NM_003299) and theamino acid sequence (GenBank® Accession No. NP 003290) of a human GRP94encoded by the DNA sequence.

SEQ ID NOs:3 and 4 are, respectively, a DNA sequence encoding a wildtype ligand binding domain of a human GRP94 and the amino acid sequenceof a human GRP94 (residues 22-337) encoded by the DNA sequence.

SEQ ID NOs:5 and 6 are, respectively, a DNA sequence encoding a ligandbinding domain of a canine GRP94 (residues 22-337) and the amino acidsequence of a canine GRP94 encoded by the DNA sequence.

SEQ ID NO:7 is peptide VSV8.

SEQ ID NO:8 is a peptide that maps to residues 271-281 of the N-terminaldomain of GRP94.

SEQ ID NO:9 is a peptide that maps to amino acids 210-222 of the humanHsp90 sequence.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is the characterization of ligand interactions ofGRP94, and where applicable Hsp90, wherein ligand binding to theN-terminal nucleotide binding domain of GRP94, and in some instances,Hsp90, elicits a conformational change that converts GRP94, and in someinstances, Hsp90, from an inactive to an active conformation, andwherein the chaperone and peptide binding activities of GRP94, and whereapplicable, Hsp90, are markedly stimulated. Also disclosed herein is thecharacterization of ligand interactions of GRP94, and where applicableHsp90, wherein ligand binding to the N-terminal nucleotide bindingdomain of GRP94, and in some instances, Hsp90, inhibits a conformationalchange that converts GRP94, and in some instances, Hsp90, from aninactive to an active conformation, and wherein the activities of GRP94,and where applicable, Hsp90, are markedly inhibited. Particularly,disclosed herein is an isolated and purified GRP94 ligand binding domain(LBD) polypeptide, and a crystalline form thereof. In a preferredembodiment, the crystalline form further comprises a ligand.

Also disclosed herein are methods of screening for ligands that bind tothe GRP94 LBD and inhibit protein activity and/or protein conformationalactivation in a manner similar and/or related to that observed withgeldanamycin and radicicol. Such ligands can function as potentialanti-tumor therapeutics, among other applications.

Until disclosure of the present invention presented herein, the abilityto obtain crystalline forms of the ligand binding domain of GRP94 hasnot been realized. And until disclosure of the present inventionpresented herein, a detailed three-dimensional crystal structure of aGRP94 LBD polypeptide has not been solved.

In addition to providing structural information, crystallinepolypeptides provide other advantages. For example, the crystallizationprocess itself further purifies the polypeptide, and satisfies one ofthe classical criteria for homogeneity. In fact, crystallizationfrequently provides unparalleled purification quality, removingimpurities that are not removed by other purification methods such asHPLC, dialysis, conventional column chromatography, and other methods.Moreover, crystalline polypeptides are sometimes stable at ambienttemperatures and free of protease contamination and other degradationassociated with solution storage. Crystalline polypeptides can also beuseful as pharmaceutical preparations. Finally, crystallizationtechniques in general are largely free of problems such as denaturationassociated with other stabilization methods (e.g., lyophilization). Oncecrystallization has been accomplished, crystallographic data providesuseful structural information that can assist the design of compoundsthat can serve as modulators (e.g. agonists or antagonists), asdescribed herein below. In addition, the crystal structure providesinformation useful to map a ligand binding domain, which can then bemimicked by a chemical entity that can serve as an antagonist oragonist.

I. Definitions

While the following terms are believed to have well defined meanings inthe art, the following definitions are set forth to facilitateexplanation of the invention.

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

As used herein the term “Hsp90 protein” is meant to refer to any of theHsp90 class of molecular chaperones that are among the most abundantproteins in eukaryotic cells, and to biologically active fragments ofsuch proteins. The term “HSP90 protein” refers to an individual memberof this class, exemplified by canine HSP90 (GENBANK® Accession No.U01153) and mouse HSP90 (SwissProt Accession No. P08113), and tobiologically active fragments thereof. Hsp90 family members arephylogenetically ubiquitous whereas the endoplasmic reticulum paralog ofHSP90, GRP94 (gp96, ERp99, endoplasmin) is found only in higher plantsand metazoans (Nicchitta (1998) Curr Opin Immunol 10:103-109). The Hsp90family of proteins are involved in directing the proper folding andtrafficking of newly synthesized proteins and in conferring protectionto the cell during conditions of heat shock, oxidative stress,hypoxic/anoxic conditions, nutrient deprivation, other physiologicalstresses, and disorders or traumas that promote such stress conditionssuch as, for example, stroke and myocardial infarction.

As used herein, the terms “ligand binding domain (LBD) of the Hsp90protein”, “Hsp90 LBD”, “GRP94 LBD”, and “HSP90 LBD” are usedinterchangeably and mean that region of an Hsp90 protein, preferably aGRP94 polypeptide or a HSP90 polypeptide, where a ligand binds. Evenmore preferably, the GRP94 LBD comprises amino acid residues 69-337 ofmammalian GRP94 (e.g., human (SEQ ID NO: 2) or canine (GENBANK®Accession No. AAA177708)), or amino acid residues 22-337 of human GRP94(which exhibits the same behavior and activity of the 69-337 LBD; seeSEQ ID NO: 4)).

As used herein, the terms “binding pocket of the GRP94 ligand bindingdomain”, “GRP94 ligand binding pocket” and “GRP94 binding pocket” areused interchangeably, and refer to the large cavity within the GRP94ligand binding domain (LBD) where a ligand can bind. This cavity can beempty, or can contain water molecules or other molecules from thesolvent, or can contain ligand atoms. The binding pocket also includesregions of space near the “main” binding pocket that not occupied byatoms of GRP94 but that are near the “main” binding pocket, and that arecontiguous with the “main” binding pocket.

“Antigenic molecule” as used herein refers to the peptides with whichGRP94 or HSP90 endogenously associates in vivo (e.g., in infected cellsor precancerous or cancerous tissue) as well as exogenousantigens/immunogens (i.e., not complexed with GRP94 or HSP90 in vivo) orantigenic/immunogenic fragments and derivatives thereof.

The term “biological activity” is meant to refer to a molecule having abiological or physiological effect in a subject. Adjuvant activity is anexample of a biological activity. Activating or inducing production ofother biological molecules having adjuvant activity is also acontemplated biological activity.

The term “adjuvant activity” is meant to refer to a molecule having theability to enhance or otherwise modulate the response of a vertebratesubject's immune system to an antigen.

The term “immune system” includes all the cells, tissues, systems,structures and processes, including non-specific and specificcategories, that provide a defense against antigenic molecules,including potential pathogens, in a vertebrate subject. As is well knownin the art, the non-specific immune system includes phagocytic cellssuch as neutrophils, monocytes, tissue macrophages, Kupffer cells,alveolar macrophages, dendritic cells and microglia. The specific immunesystem refers to the cells and other structures that impart specificimmunity within a host. Included among these cells are the lymphocytes,particularly the B cell lymphocytes and the T cell lymphocytes. Thesecells also include natural killer (NK) cells. Additionally,antibody-producing cells, like B lymphocytes, and the antibodiesproduced by the antibody-producing cells are also included within theterm “immune system”.

The term “immune response” is meant to refer to any response to anantigen or antigenic determinant by the immune system of a vertebratesubject. Exemplary immune responses include humoral immune responses(e.g. production of antigen-specific antibodies) and cell-mediatedimmune responses (e.g. lymphocyte proliferation), as defined hereinbelow.

The term “systemic immune response” is meant to refer to an immuneresponse in the lymph node-, spleen-, or gut-associated lymphoid tissueswherein cells, such as B lymphocytes, of the immune system aredeveloped. For example, a systemic immune response can comprise theproduction of serum IgG's. Further, systemic immune response refers toantigen-specific antibodies circulating in the blood stream andantigen-specific cells in lymphoid tissue in systemic compartments suchas the spleen and lymph nodes.

The terms “humoral immunity” or “humoral immune response” are meant torefer to the form of acquired immunity in which antibody molecules aresecreted in response to antigenic stimulation.

The terms “cell-mediated immunity” and “cell-mediated immune response”are meant to refer to the immunological defense provided by lymphocytes,such as that defense provided by T cell lymphocytes when they come intoclose proximity to their victim cells. A cell-mediated immune responsealso comprises lymphocyte proliferation. When “lymphocyte proliferation”is measured, the ability of lymphocytes to proliferate in response tospecific antigen is measured. Lymphocyte proliferation is meant to referto B cell, T-helper cell or CTL cell proliferation.

The term “CTL response” is meant to refer to the ability of anantigen-specific cell to lyse and kill a cell expressing the specificantigen. As described herein below, standard, art-recognized CTL assaysare performed to measure CTL activity.

“Adoptive immunotherapy” as used herein refers to a therapeutic approachwith particular applicability to cancer whereby immune cells with anantitumor reactivity are administered to a tumor-bearing host, with theaim that the cells mediate either directly or indirectly, the regressionof an established tumor.

An “immunogenic composition” is meant to refer to a composition that canelicit an immune response. A vaccine is contemplated to fall within themeaning of the term “immunogenic composition”, in accordance with thepresent invention.

The term “a biological response modifier” is meant to refer to amolecule having the ability to enhance or otherwise modulate a subject'sresponse to a particular stimulus, such as presentation of an antigen.

As used herein, the terms “candidate substance” and “candidate compound”are used interchangeably and refer to a substance that is believed tointeract with another moiety as a biological response modifier. Forexample, a representative candidate compound is believed to interactwith a complete Hsp90 protein, or fragment thereof, and which can besubsequently evaluated for such an interaction. Exemplary candidatecompounds that can be investigated using the methods of the presentinvention include, but are not restricted to, agonists and antagonistsof an Hsp90 protein, viral epitopes, peptides, enzymes, enzymesubstrates, co-factors, lectins, sugars, oligonucleotides or nucleicacids, oligosaccharides, proteins, chemical compounds small molecules,and monoclonal antibodies.

As used herein, the term “modulate” means an increase, decrease, orother alteration of any or all chemical and biological activities orproperties of a wild-type or mutant Hsp90 protein, preferably awild-type or mutant GRP94 or HSP90 polypeptide. The term “modulation” asused herein refers to both upregulation (i.e., activation orstimulation) and downregulation (i.e. inhibition or suppression) of aresponse.

As used herein, the term “agonist” means an agent that supplements orpotentiates the biological activity of a functional Hsp90 protein.

As used herein, the term “antagonist” means an agent that decreases orinhibits the biological activity of a functional Hsp90 protein, or thatsupplements or potentiates the biological activity of a naturallyoccurring or engineered non-functional Hsp90 protein.

As used herein, the terms “α-helix”, “alpha-helix” and “alpha helix” areused interchangeably and mean the conformation of a polypeptide chainwherein the polypeptide backbone is wound around the long axis of themolecule in a left-handed or right-handed direction, and the R groups ofthe amino acids protrude outward from the helical backbone, wherein therepeating unit of the structure is a single turnoff the helix, whichextends about 0.56 nm along the long axis.

As used herein, the terms “β-sheet”, “beta-sheet” and “beta sheet” areused interchangeably and mean the conformation of a polypeptide chainstretched into an extended zig-zig conformation. Portions of polypeptidechains that run “parallel” all run in the same direction. Polypeptidechains that are “antiparallel” run in the opposite direction from theparallel chains.

As used herein, the term “crystal lattice” means the array of pointsdefined by the vertices of packed unit cells.

As used herein, the term “unit cell” means a basic parallelipiped shapedblock. The entire volume of a crystal can be constructed by regularassembly of such blocks. Each unit cell comprises a completerepresentation of the unit of pattern, the repetition of which builds upthe crystal. Thus, the term “unit cell” means the fundamental portion ofa crystal structure that is repeated infinitely by translation in threedimensions. A unit cell is characterized by three vectors a, b, and c,not located in one plane, which form the edges of a parallelepiped.Angles α, β, and γ define the angles between the vectors: angle α is theangle between vectors b and c; angle β is the angle between vectors aand c; and angle γ is the angle between vectors a and b. The entirevolume of a crystal can be constructed by regular assembly of unitcells; each unit cell comprises a complete representation of the unit ofpattern, the repetition of which builds up the crystal.

As used herein, “orthorhombic unit cell” means a unit cell whereina≠b≠c; and α=β=γ=90°. The vectors a, b and c describe the unit celledges and the angles α, β, and γ describe the unit cell angles. In afirst preferred embodiment of the present invention, the unit cell haslattice constants of a=99.889 Å, b=89.614 Å, c=60.066 Å, β=90.132°. In asecond preferred embodiment of the present invention, the unit cell haslattice constants of the unit cell has lattice constants of a=89.200 Å,b=99.180 Å, c=63.071 Å; α=β=γ=90.0°. While preferred lattice constantsare provided, a crystalline polypeptide of the present invention alsocomprises variations from the preferred lattice constants, wherein thevariations range from about one to about two percent.

As used herein, the terms “cells,” “host cells” or “recombinant hostcells” are used interchangeably and mean not only to the particularsubject cell, but also to the progeny or potential progeny of such acell. Because certain modifications can occur in succeeding generationsdue to either mutation or environmental influences, such progeny mightnot, in fact, be identical to the parent cell, but are still includedwithin the scope of the term as used herein.

As used herein, the terms “chimeric protein” or “fusion protein” areused interchangeably and mean a fusion of a first amino acid sequenceencoding an Hsp90 polypeptide with a second amino acid sequence defininga polypeptide domain foreign to, and not homologous with, any domain ofa. Hsp90 polypeptide (preferably a GRP94 polypeptide). For example, achimeric protein can include a foreign domain that is found in anorganism that also expresses the first protein, or it can be an“interspecies” or “intergenic” fusion of protein structures expressed bydifferent kinds of organisms. In general, a fusion protein can berepresented by the general formula X-GRP94-Y, wherein GRP94 represents aportion of the protein which is derived from a GRP94 polypeptide, and Xand Y are independently absent or represent amino acid sequences whichare not related to a GRP94 sequence in an organism, which includesnaturally occurring mutants.

As used herein, the term “detecting” means confirming the presence of atarget entity by observing the occurrence of a detectable signal, suchas a radiologic or spectroscopic signal that will appear exclusively inthe presence of the target entity.

As used herein, the term “DNA segment” means a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species. In apreferred embodiment, a DNA segment encoding a GRP94 polypeptide refersto a DNA segment that comprises any of SEQ ID NOs:1, 3 or 5, but canoptionally comprise fewer or additional nucleic acids, yet is isolatedaway from, or purified free from, total genomic DNA of a source species,such as Homo sapiens. Included within the term “DNA segment” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phages, viruses, andthe like.

As used herein, the term “DNA sequence encoding a GRP94 polypeptide” canrefer to one or more coding sequences within a particular individual.Moreover, certain differences in nucleotide sequences can exist betweenindividual organisms, which are called alleles. It is possible that suchallelic differences might or might not result in differences in aminoacid sequence of the encoded polypeptide yet still encode a protein withthe same biological activity. As is well known, genes for a particularpolypeptide can exist in single or multiple copies within the genome ofan individual. Such duplicate genes can be identical or can have certainmodifications, including nucleotide substitutions, additions ordeletions, all of which still code for polypeptides having substantiallythe same activity.

As used herein, the terms “GRP94 gene product”, “GRP94 protein”, “GRP94polypeptide”, and “GRP94 peptide” are used interchangeably and meanpeptides having amino acid sequences which are substantially identicalto native amino acid sequences from the organism of interest and whichare biologically active in that they comprise all or a part of the aminoacid sequence of a GRP94 polypeptide, or cross-react with antibodiesraised against a GRP94 polypeptide, or retain all or some of thebiological activity (e.g., DNA or ligand binding ability and/ortranscriptional regulation) of the native amino acid sequence orprotein. Such biological activity can include immunogenicity. Apreferred embodiment in a GRP94 LBD polypeptide, and representativeembodiments of a GRP94 LBD are set forth in SEQ ID NOs:4 and 6.

The terms “GRP94 gene product”, “GRP94 protein”, “GRP94 polypeptide”,and “GRP94 peptide” also include analogs of a GRP94 polypeptide. By“analog” is intended that a DNA or peptide sequence can containalterations relative to the sequences disclosed herein, yet retain allor some of the biological activity of those sequences. Analogs can bederived from nucleotide sequences as are disclosed herein or from otherorganisms, or can be created synthetically. Those skilled in the artwill appreciate that other analogs, as yet undisclosed or undiscovered,can be used to design and/or construct GRP94 analogs. There is no needfor a “GRP94 gene product”, “GRP94 protein”, “GRP94 polypeptide”, or“GRP94 peptide” to comprise all or substantially all of the amino acidsequence of a GRP94 gene product. Shorter or longer sequences areanticipated to be of use in the invention; shorter sequences are hereinreferred to as “segments”. Thus, the terms “GRP94 gene product”, “GRP94protein”, “GRP94 polypeptide”, and “GRP94 peptide” also include fusionor recombinant GRP94 polypeptides and proteins comprising sequences ofthe present invention. Methods of preparing such proteins are disclosedherein and are known in the art.

As used herein, the terms “GRP94 gene” and “recombinant GRP94 gene” meana nucleic acid molecule comprising an open reading frame encoding aGRP94 polypeptide of the present invention, including both exon and(optionally) intron sequences.

As used herein, the term “interact” means detectable interactionsbetween molecules, such as can be detected using, for example, a yeasttwo-hybrid assay. The term “interact” is also meant to include “binding”interactions between molecules. Interactions can, for example, beprotein-protein or protein-nucleic acid in nature.

As used herein, the term “labeled” means the attachment of a moiety,capable of detection by spectroscopic, radiologic or other methods, to aprobe molecule.

As used herein, the term “modified” means an alteration from an entity'snormally occurring state. An entity can be modified by removing discretechemical units or by adding discrete chemical units. The term “modified”encompasses detectable labels as well as those entities added as aids inpurification.

As used herein, the term “molecular replacement” means a method thatinvolves generating a preliminary model of the wild-type GRP94 ligandbinding domain, or a GRP94 mutant crystal whose structure coordinatesare unknown, by orienting and positioning a molecule or model whosestructure coordinates are known (e.g., an Hsp) within the unit cell ofthe unknown crystal so as best to account for the observed diffractionpattern of the unknown crystal. Phases can then be calculated from thismodel and combined with the observed amplitudes to give an approximateFourier synthesis of the structure whose coordinates are unknown. This,in turn, can be subject to any of the several forms of refinement toprovide a final, accurate structure of the unknown crystal. See, e.g.,Lattman, (1985) Method Enzymol., 115: 55-77; Rossmann, ed, (1972) TheMolecular Replacement Method, Gordon & Breach, New York. Using thestructure coordinates of the ligand binding domain of GRP94 provided bythis invention, molecular replacement can be used to determine thestructure coordinates of a crystalline mutant or homologue (e.g.,another Hsp90 polypeptide or Hsp90 LBD polypeptide) of the GRP94 ligandbinding domain, or of a different crystal form of the GRP94 ligandbinding domain.

As used herein, the term “mutation” carries its traditional connotationand means a change, inherited, naturally occurring or introduced, in anucleic acid or polypeptide sequence, and is used in its sense asgenerally known to those of skill in the art.

As used herein, the term “partial agonist” means an entity that can bindto a target and induce only part of the changes in the target that areinduced by agonists. The differences can be qualitative or quantitative.Thus, a partial agonist can induce some of the conformation changesinduced by agonists, but not others, or it can only induce certainchanges to a limited extent.

As used herein, the term “partial antagonist” means an entity that canbind to a target and inhibit only part of the changes in the target thatare induced by antagonists. The differences can be qualitative orquantitative. Thus, a partial antagonist can inhibit some of theconformation changes induced by an antagonist, but not others, or it caninhibit certain changes to a limited extent.

As used herein, the term “polypeptide” means any polymer comprising anyof the 20 protein amino acids, regardless of its size. Although“protein” is often used in reference to relatively large polypeptides,and “peptide” is often used in reference to small polypeptides, usage ofthese terms in the art overlaps and varies. The term “polypeptide” asused herein refers to peptides, polypeptides and proteins, unlessotherwise noted. As used herein, the terms “protein”, “polypeptide” and“peptide” are used interchangeably herein when referring to a geneproduct.

As used herein, the term “sequencing” means the determining the orderedlinear sequence of nucleic acids or amino acids of a DNA or proteintarget sample, using conventional manual or automated laboratorytechniques.

As used herein, the term “space group” means the arrangement of symmetryelements of a crystal.

As used herein, the terms “structure coordinates” and “structuralcoordinates” mean mathematical coordinates derived from mathematicalequations related to the patterns obtained on diffraction of amonochromatic beam of X-rays by the atoms (scattering centers) of amolecule in crystal form. The diffraction data are used to calculate anelectron density map of the repeating unit of the crystal. The electrondensity maps are used to establish the positions of the individual atomswithin the unit cell of the crystal.

Those of skill in the art understand that a set of coordinatesdetermined by X-ray crystallography is not without standard error. Ingeneral, the error in the coordinates tends to be reduced as theresolution is increased, since more experimental diffraction data isavailable for the model fitting and refinement. Thus, for example, morediffraction data can be collected from a crystal that diffracts to aresolution of 2.8 angstroms than from a crystal that diffracts to alower resolution, such as 3.5 angstroms. Consequently, the refinedstructural coordinates will usually be more accurate when fitted andrefined using data from a crystal that diffracts to higher resolution.The design of ligands and modulators for GRP94 or any other Hsp90polypeptide depends on the accuracy of the structural coordinates. Ifthe coordinates are not sufficiently accurate, then the design processwill be ineffective. In most cases, it is very difficult or impossibleto collect sufficient diffraction data to define atomic coordinatesprecisely when the crystals diffract to a resolution of only 3.5angstroms or poorer. Thus, in most cases, it is difficult to use X-raystructures in structure-based ligand design when the X-ray structuresare based on crystals that diffract to a resolution of only 3.5angstroms or poorer. However, common experience has shown that crystalsdiffracting to 2.8 angstroms or better can yield X-ray structures withsufficient accuracy to greatly facilitate structure-based drug design.Further improvement in the resolution can further facilitatestructure-based design, but the coordinates obtained at 2.8 angstromsresolution are generally adequate for most purposes.

Also, those of skill in the art will understand that Hsp90 proteins canadopt different conformations when different ligands are bound. Inparticular, Hsp90 proteins will adopt substantially differentconformations when agonists and antagonists are bound. Subtle variationsin the conformation can also occur when different agonists are bound,and when different antagonists are bound. These variations can bedifficult or impossible to predict from a single X-ray structure.Generally, structure-based design of GRP94 modulators depends to somedegree on knowledge of the differences in conformation that occur whenagonists and antagonists are bound. Thus, structure-based modulatordesign is most facilitated by the availability of X-ray structures ofcomplexes with potent agonists as well as potent antagonists.

As used herein, the term “target cell” refers to a cell, into which itis desired to insert a nucleic acid sequence or polypeptide, or tootherwise effect a modification from conditions known to be standard inthe unmodified cell. A nucleic acid sequence introduced into a targetcell can be of variable length. Additionally, a nucleic acid sequencecan enter a target cell as a component of a plasmid or other vector oras a naked sequence.

II. Description of Tables

Table 1 is a table of the atomic structure coordinate data obtained fromX-ray diffraction from Form 1 of the ligand binding domain of canineGRP94 (residues 69-337337 of GENBANK® Accession No. AAA17708; residues48-316 of SEQ ID NO: 6) in complex with NECA.

Table 2 is a table of the atomic structure coordinates of the NECA andGRP94 binding pocket within Form 1 of the canine GRP94 LBD.

Table 3 is a table of the atomic structure coordinate data obtained fromX-ray diffraction from Form 2 of the ligand binding domain of canineGRP94 (residues 69-337337 of GENBANK® Accession No. AAA17708; residues48-316 of SEQ ID NO: 6) in complex with NECA.

III. General Considerations

As noted above, GRP94 (gp96, ERp99, endoplasmin) is the endoplasmicreticulum paralog of cytosolic HSP90, and as such, is an abundantresident ER lumenal protein that by virtue of its association withnascent polypeptides performs a chaperone function. The terms “GRP94”“GRP94 polypeptide”, and/or “GRP94 protein” also refer to biologicallyactive fragments of a GRP94 protein. A preferred biologically activefragment is the GRP94 LBD. Consistent with this role, GRP94 expressionis upregulated by stress conditions that promote protein misfolding orunfolding, such as glucose starvation, oxidative stress, and heavy metalpoisoning. In addition to its role in the regulation of protein foldingin the ER, GRP94 can function in the intercellular trafficking ofpeptides from the extracellular space to the major histocompatabilitycomplex (MHC) class I antigen processing pathway of professional antigenpresenting cells. Thus, in addition to a homeostatic role in proteinfolding and assembly, GRP94 functions as a component of the MHC class Iantigen processing and presentation pathways of mammalian cells.

GRP94 also contributes to the folding and assembly of immunoglobulins,MHC class II molecules, HSV-1 glycoproteins, thyroglobulin, collagen,and p185erbB2. (Melnick et al. (1992) J Biol Chem 267:21303-21306;Melnick et al. (1994) Nature 370:373-375; Schaiff et al. (1992) J ExpMed 176:657-666; Navarro et al. (1991) Virology 184:253-264; Kuznetsovet al. (1994) J Biol Chem 269:22990-22995; Ferreira et al. (1994) J CellBiochem 56:518-26; Chavany et al. (1996) J Biol Chem 273:4974-4977). Inaddition to interactions with polypeptide folding substrates, GRP94binds peptides, a subset of which is suitable for assembly on nascentMHC class I molecules. (Srivastava et al. (1986) Proc Natl Acad Sci USA83:3407-3411; Nieland et al. (1996) Proc Natl Acad Sci USA 93:6135-6139;Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156; Ishii et al.(1999) J Immunol 162:1303-1309; Srivastava et al. (1998) Immunity8:657-665; Sastry & Linderoth (1999) J Biol Chem 274:12023-12035). Thepeptide binding activity of GRP94 plays a role in its ability to elicitCD8+T cell immune responses. (Udono et al. (1994) Proc Natl Acad SciUSA, 91:3077-30781; Suto & Srivastava (1995) Science 269:1585-1588;Arnold et al. (1995) J Exp Med 182:885-889; Nair et al. (1999) J Immunol162:6426-6432; Blachere et al. (1997) J Exp Med 186:465-472; Heike etal. (1996) J Leukoc Biol 139:613-623; Srivastava et al. (1998) Immunity8:657-665). Peptide binding activity is not, however, alone sufficientto impart immunogenic activity to a protein and thus GRP94 is among alimited subset of molecular chaperones that can function in theessential immunological process of cross-presentation. (Srivastava etal. (1998) Immunity 8:657-665; Nair et al. (1999) J Immunol162:6426-6432; Basu and Srivastava (1999) J Exp Med 189:797-802; Schildet al. (1999) Curr Opin Immunol 11:109-113).

HSP90 has adenosine nucleotide-dependent modes of regulation.Additionally, amino acid side chains that participate in water-mediatedhydrogen bonds with the N7 group of the purine ring of adenosine (N51 inhuman HSP90=N86 in GRP94) and the N1 group of the purine ring ofadenosine (G97 in human HSP90=G130 of GRP94) are conserved between HSP90and GRP94. The N6 group of the purine ring of adenosine (L48 in humanHSP90=L83 in GRP94) that mediates direct nucleotide binding is alsoconserved between HSP90 and GRP94. In ATP binding with HSP90, the N6group of the adenine purine is the sole direct hydrogen bond between thenucleotide and the nucleotide binding pocket (Prodromou et al. (1997)Cell 90:65-75; Obermann et al. (1998) J Cell Biol 143:901-910), and N6substituted adenosine analogs do not bind to GRP94. (Hutchison & Fox(1989) J Biol Chem 264:19898-903; Hutchison et al. (1990) Biochemistry29:5138-5144). Thus, although ATP/ADP binding and hydrolysis aregenerally accepted as biological properties of HSP90, it is not knownwhether ATP/ADP serve an identical function(s) in the regulation ofGRP94 activity. ATP and ADP bind with very low affinity to GRP94 andthus experimental limitations require that ATP/ADP interactions at theGRP94 nucleotide binding pocket be analyzed by indirect methods,including but not limited to ligand displacement assays. (Wearsch et al.(1998) Biochemistry 37(16):5709-5719; Csermely et al. (1995) J Biol Chem270:6381-6388; Li & Srivastava (1993) EMBO J 12:3143-3151).

The peptide binding activity of GRP94 plays a role in its ability toelicit CD8⁺ T cell immune responses. Peptide binding activity is not,however, alone sufficient to impart immunogenic activity to a proteinand thus GRP94 is among a limited subset of molecular chaperones thatcan function in the essential immunological process ofcross-presentation. Until the disclosure of the present invention, acrystalline form providing a detailed look at a GRP94 ligand-interactionthat modulates activity of GRP94 with respect to both polypeptide andpeptide substrates remained to be determined.

HSP90 and GRP94 have been proposed as possible targets of severalantitumor agents, principally radicicol and geldanamycin. Scheibel &Buckner (1998) Biochem Pharm 56:675-82. These compounds are believed toact by inhibiting the ability of the Hsp90 proteins to assistproto-oncogenic kinases, hormone receptors, and other signaling proteinsassume their active folded states and appropriate subcellular location.Pratt (1998) Proc Soc Exp Biol Med 217:420-434.

GRP94 has also been found to elicit cytotoxic T cell responses, areflection of its peptide binding activity (Nicchitta (1998) Curr OpinImmunol 10:103-109; Srivastava et al. (1998) Immunity 8:657-665). It isnow established that GRP94-peptide complexes can be processed byprofessional antigen presenting cells, with the GRP94-bound peptidesexchanged onto MHC class I molecules of the antigen presenting cell. Theantigen presenting cells can then interact with naive CD8⁺ T cellresponses against tissue(s) displaying peptide epitopes present incomplex with GRP94 (Srivastava et al. (1998) Immunity 8:657-665).

A potential yet heretofore uncharacterized protective role of grp94 inischemia is supported by the observation that expression of GRP94 isenhanced in hippocampus after transient forebrain ischemia of a durationknown to result in neuronal death (Yagita et al. (1999) J Neurochem72:1544-1551). grp94 is similarly induced following acute kidneyischemia (Kuznetsov (1996) Proc Natl Acad Sci USA 93:8584-8589).Heat-shock proteins, including HSP90, are overexpressed during theoxidative stress of reperfusion that generally follows ischemia(Sciandra et al. (1984) Proc Natl Acad Sci USA 81:4843-4847). HSP90might also play a role in ischemic signaling by binding to thehypoxia-inducible factor 1-a (Gradin et al. (1996) Mol Cell Biol16:5221-5231).

Summarily, in accordance with the present invention, GRP94 and HSP90represent rational targets for chemotherapeutics, immunotherapeutics andvaccines relevant to the treatment of infections disease and cancer. Inview of their function as molecular chaperones, GRP94 and HSP90 furtherrepresent rational targets for the development of therapeutics fortissue injury and stress, such as can occur in ischemic injuriesincluding, but not limited to, organ (kidney, heart, lung, liver)transplantation, cerebral stroke, and myocardial infarct. Furthermore,Hsp90 and GRP94 represent rational targets for anti-tumor drug design.

Despite the aforementioned indirect characterization of the structure ofGRP94, until the present disclosure, a detailed three-dimensional modelof GRP94, including particularly the ligand binding domain of GRP94, hasnot been achieved.

Sequence analysis and X-ray crystallography, including the disclosure ofthe present invention, have confirmed that GRP94 has a modulararchitecture, with three domains, including a N-terminal ligand bindingdomain (LBD). The modularity of GRP94 permits different domains of eachprotein to separately accomplish certain functions. Some of thefunctions of a domain within the full-length protein are preserved whenthat particular domain is isolated from the remainder of the protein.Using conventional protein chemistry techniques, a modular domain cansometimes be separated from the parent protein. Using conventionalmolecular biology techniques, each domain can usually be separatelyexpressed with its original function intact or, as discussed hereinbelow, chimeras comprising two different proteins can be constructed,wherein the chimeras retain the properties of the individual functionaldomains of the protein from which the chimeras were generated.

As described herein, the LBD of a GRP94 can be mutated or engineered,expressed, crystallized, its three dimensional structure determined witha ligand bound as disclosed in the present invention, and computationalmethods can be used to design ligands to heat shock proteins, preferablyto Hsp90 proteins, and more preferably to GRP94. Thus, the presentinvention will usually be applicable mutatis mutandis to heat shockproteins, more particularly to Hsp90 proteins and even more particularlyto GRP94 proteins, including GRP94 isoforms, as discussed herein, based,in part, on the patterns of heat shock protein structure and modulationthat have emerged as a consequence of the present disclosure, which inpart discloses determining the three dimensional structure of the ligandbinding domain of GRP94 in complex with NECA.

IV. The NECA Liqand

Ligand binding can induce biological functions in a variety of ways. Inone aspect of the present invention, a GRP94 LBD is co-crystallized witha ligand, preferably a ligand that bears structural similarities to thepurine nucleoside, adenosine. In a preferred embodiment, a crystallineform of the present invention comprises a GRP94 LBD in complex withN-ethylcarboxamidoadenosine (NECA). A crystalline form of the presentinvention can also comprise a ligand composition of the presentinvention as disclosed herein below. Thus, an aspect of the presentinvention is the definition of the protein fold and space occupied bythe ligand provided by the crystalline form.

V. Atomic Structure of Hsp90 Polypeptides

A Hsp90 protein can be expressed, crystallized, its three dimensionalstructure determined with a ligand bound as disclosed in the presentinvention. Once crystallization has been accomplished, crystallographicdata provides useful structural information that can assist the designof compounds that function as agonists or antagonists, as describedherein below. In a preferred embodiment, the Hsp90 polypeptide is GRP94or a fragment thereof.

The crystallization process also purifies the polypeptide, and satisfiesone of the classical criteria for homogeneity. In fact, crystallizationfrequently provides unparalleled purification quality, removingimpurities that are not removed by other purification methods such asHPLC, dialysis, conventional column chromatography, etc. Moreover,crystalline polypeptides are often stable at ambient temperatures andfree of protease contamination and other degradation associated withsolution storage. Crystallization techniques in general yield a purepolypeptide largely free of problems such as denaturation associatedwith other stabilization methods (e.g., lyophilization such crystallizedpolypeptides can be useful as pharmaceutical preparations).

V.A. Production of Hsp90 Polypeptides

According to the present invention, an Hsp90 polypeptide, preferably aGRP94 or GRP94 LBD polypeptide, can be expressed using an expressionvector. An expression vector, as is well known to those of skill in theart, typically includes elements that permit autonomous replication in ahost cell independent of the host genome, and one or more phenotypicmarkers for selection purposes. Either prior to or after insertion ofthe DNA sequences surrounding the desired Hsp90 or GRP94 (e.g., GRP94LBD) coding sequence, an expression vector also will include controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes and a signal for termination. In someembodiments, where secretion of the produced polypeptide is desired,nucleotides encoding a “signal sequence” can be inserted prior to anHsp90 or GRP94, or Hsp90 or GRP94 LBD, coding sequence. For expressionunder the direction of the control sequences, a desired DNA sequencemust be operatively linked to the control sequences; that is, thesequence must have an appropriate start signal in front of the DNAsequence encoding the Hsp90 or GRP94, or Hsp90 or GRP94 LBD polypeptide,and the correct reading frame to permit expression of that sequenceunder the control of the control sequences and production of the desiredproduct encoded by that Hsp90 or GRP94, or Hsp90 or GRP94 LBD, sequencemust be maintained.

After a review of the disclosure of the present invention presentedherein, any of a wide variety of well-known available expression vectorscan be useful to express a mutated coding sequence of this invention.These include for example, vectors consisting of segments ofchromosomal, non-chromosomal and synthetic DNA sequences, such asvarious known derivatives of SV40, known bacterial plasmids, e.g.,plasmids from E. coli including col E1, pCR1, pBR322, pMB9 and theirderivatives, wider host range plasmids, e.g., RP4, phage DNAs, e.g., thenumerous derivatives of phage λ, e.g., NM 989, and other DNA phages,e.g., M13 and filamentous single stranded DNA phages, yeast plasmids andvectors derived from combinations of plasmids and phage DNAs, such asplasmids which have been modified to employ phage DNA or otherexpression control sequences.

In addition, any of a wide variety of expression controlsequences—sequences that control the expression of a DNA sequence whenoperatively linked to it—can be used in these vectors to express themutated DNA sequences according to this invention. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40 for animal cells, the lac system, the trp system theTAC or TRC system, the major operator and promoter regions of phage λ,the control regions of fd coat protein, all for E. coli, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors for yeast, and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof.

A wide variety of hosts are also useful for producing Hsp90 or GRP94, orHsp90 or GRP94 LBD, polypeptides according to this invention. Thesehosts include, for example, bacteria, such as E. coli, Bacillus andStreptomyces, fungi, such as yeasts, and animal cells, such as CHO andCOS-1 cells, plant cells, insect cells, such as SF9 cells, andtransgenic host cells.

It should be understood that not all expression vectors and expressionsystems function in the same way to express DNA sequences of thisinvention, and to produce Hsp90 or GRP94 polypeptide, Hsp90 or GRP94 LBDpolypeptides, Hsp90 or GRP94 mutants, or Hsp90 or GRP94 LBD mutants.Neither do all hosts function equally well with the same expressionsystem. One of skill in the art can, however, make a selection amongthese vectors, expression control sequences and hosts without undueexperimentation and without departing from the scope of this invention.For example, an important consideration in selecting a vector will bethe ability of the vector to replicate in a given host. The copy numberof the vector, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe system, its controllability and its compatibility with the DNAsequence encoding an Hsp90 or GRP94 polypeptide, or Hsp90 or GRP94 LBDpolypeptide of this invention, with particular regard to the formationof potential secondary and tertiary structures.

Hosts should be selected by consideration of their compatibility withthe chosen vector, the toxicity of a modified polypeptide to them, theirability to express mature products, their ability to fold proteinscorrectly, their fermentation requirements, the ease of purification ofan Hsp90 or GRP94, or Hsp90 or GRP94 LBD, and safety. Within theseparameters, one of skill in the art can select various vector/expressioncontrol system/host combinations that will produce useful amounts of apolypeptide. A polypeptide produced in these systems can be purified,for example, via the approaches disclosed in the Examples.

Thus, it is envisioned, based upon the disclosure of the presentinvention, that purification of the unliganded or liganded Hsp90 orGRP94, or Hsp90 or GRP94 LBD, polypeptide can be obtained byconventional techniques, such as hydrophobic interaction chromatography(HPLC), ion exchange chromatography (HPLC), gel filtrationchromatography, and heparin affinity chromatography. To achieve higherpurification for improved crystals Hsp90 or GRP94, or Hsp90 or GRP94LBD, polypeptide it is sometimes preferable to ligand shift purify theHsp90 or GRP94, or Hsp90 or GRP94 LBD, polypeptide using a column thatseparates the polypeptide according to charge, such as an ion exchangeor hydrophobic interaction column, and then bind the eluted polypeptidewith a ligand. The ligand induces a change in the polypeptide's surfacecharge such that when re-chromatographed on the same column, thepolypeptide then elutes at the position of the liganded polypeptide andis removed by the original column run with the unliganded polypeptide.Typically, saturating concentrations of ligand can be used in the columnand the polypeptide can be preincubated with the ligand prior to passingit over the column.

More recently developed methods involve engineering a “tag” such as withhistidine placed on the end of the protein, such as on the aminoterminus, and then using a nickel chelation column for purification. SeeJanknecht, (1991) Proc. Natl. Acad. Sci. U.S.A. 88: 8972-8976 (1991),incorporated by reference.

In a preferred embodiment as disclosed in the Examples, canine GRP94 LBD(residues 69-337) of GENBANK® Accession No. AAA17708; residues 48-316 ofSEQ ID NO: 6) was overexpressed as a GST fusion in E. coli and purifiedto homogeneity by affinity and ion-exchange chromatography. The proteinwas exchanged into 10 mM Tris-HCl, pH 7.6, 1 mM DTT, 100 mM NaCl andconcentrated to 30 mg/mL.

V.B. Formation of Hsp90 Protein Crystals

In one embodiment, the present invention provides crystals of a GRP94ligand binding domain (LBD) polypeptide. A preferred approach forobtaining the crystals is disclosed in the Examples. The GRP94 LBDcrystals, which can be native crystals, derivative crystals orco-crystals, have c-centered orthorhombic unit cells. In a firstpreferred embodiment of the present invention, the unit cell has latticeconstants of a=99.889 Å, b=89.614 Å, c=60.066 Å; β=90.132°; space groupsymmetry C2; and 2 GRP94+NECA complexes in the asymmetric unit. In asecond preferred embodiment of the present invention, the unit cell haslattice constants of a=89.200 Å, b=99.180 Å, c=63.071 Å; α=β=γ=90.0°;space group symmetry C222₍₁₎; and 1 GRP94+NECA complex in the asymmetricunit.

The native and derivative co-crystals, and fragments thereof, disclosedin the present invention can be obtained by a variety of techniques,including batch, liquid bridge, dialysis, vapor diffusion and hangingdrop methods. See, e.g., McPherson (1982) Preparation and Analysis ofProtein Crystals, John Wiley, New York, N.Y.; McPherson (1990) Eur JBiochem 189:1-23; Weber (1991) Adv Protein Chem 41:1-36). In a preferredembodiment, hanging drop methods are used for the crystallization ofHsp90 proteins and fragments thereof. A more preferred hanging dropmethod is disclosed in the Examples.

In general, native crystals of the present invention are grown bydissolving a substantially pure Hsp90 protein or a fragment thereof inan aqueous buffer containing a precipitant at a concentration just belowthat necessary to precipitate the protein. Water is removed bycontrolled evaporation to produce precipitating conditions, which aremaintained until crystal growth ceases.

In one embodiment of the invention, native crystals are grown by vapordiffusion (See, e.g., McPherson, (1982) Preparation and Analysis ofProtein Crystals, John Wiley, New York.; McPherson, (1990) Eur. J.Biochem. 189:1-23). In this method, the polypeptide/precipitant solutionis allowed to equilibrate in a closed container with a larger aqueousreservoir having a precipitant concentration optimal for producingcrystals. Generally, less than about 25 μL of Hsp90 (preferably GRP94LBD) polypeptide solution is mixed with an equal volume of reservoirsolution, giving a precipitant concentration about half that requiredfor crystallization. This solution is suspended as a droplet underneatha coverslip, which is sealed onto the top of the reservoir. The sealedcontainer is allowed to stand, until crystals grow. Crystals generallyform within two to six weeks, and are suitable for data collectionwithin approximately seven to ten weeks. Of course, those of skill inthe art will recognize that the above-described crystallizationprocedures and conditions can be varied.

V.C. Preparation of Derivative Crystals

Derivative crystals of the present invention, e.g. heavy atom derivativecrystals, can be obtained by soaking native crystals in mother liquorcontaining salts of heavy metal atoms. Such derivative crystals areuseful for phase analysis in the solution of crystals of the presentinvention. In a preferred embodiment of the present invention, forexample, growing the GRP94 overexpressing bacteria in minimal mediacontaining Se-Met provides protein containing Se-Met in place of Met andprovides crystals suitable for use in multi-wavelength anomalousdispersion X-ray phasing methods. Additional reagents useful for thepreparation of the derivative crystals of the present invention will beapparent to those of skill in the art after review of the disclosure ofthe present invention.

V.D. Preparation of Co-crystals

Co-crystals of the present invention can be obtained by soaking a nativecrystal in mother liquor containing compounds known or predicted to bindthe Hsp90 protein, or a fragment thereof. Alternatively, co-crystals canbe obtained by co-crystallizing a Hsp90 protein or a fragment thereof inthe presence of one or more compounds known or predicted to bind thepolypeptide. In a preferred embodiment, such a compound is NECA, and theHsp90 protein is GRP94 or a fragment thereof.

V.E. Solving a Crystal Structure of the Present Invention

Crystal structures of the present invention can be solved using avariety of techniques including, but not limited to, isomorphousreplacement anomalous scattering or molecular replacement methods.Computer software packages will also be helpful in solving a crystalstructure of the present invention. Applicable software packages includebut are not limited to X-PLOR™ program (Brunger (1992) X-PLOR, Version3.1. A System for X-ray Crystallography and NMR, Yale University Press,New Haven, Conn.; X-PLOR is available from Molecular Simulations, Inc.,San Diego, Calif.), Xtal View (McRee (1992) J Mol Graphics 10:44-47;X-tal View is available from the San Diego Supercomputer Center), SHELXS97 (Sheldrick (1990) Acta Cryst A46:467; SHELX 97 is available from theInstitute of Inorganic Chemistry, Georg-August-Universität, Gottingen,Germany), HEAVY (Terwilliger, Los Alamos National Laboratory) andSHAKE-AND-BAKE (Hauptman (1997) Curr Opin Struct Biol 7:672-680; Weekset al. (1993) Acta Cryst D49:179; available from the Hauptman-WoodwardMedical Research Institute, Buffalo, N.Y.) can be used. See also,Ducruix & Geige (1992) Crystallization of Nucleic Acids and Proteins: APractical Approach, IRL Press, Oxford, England, and references citedtherein.

V.F. Characterization of the GRP94 LBD Crystal

The following amino acid residues of the GRP94 LBD form the pocket intowhich NECA binds and forms specific interactions with the protein: Met85, Glu 103, Leu 104, Asn 107, Ala 108, Asp 110, Ala 111, Val 147, Asp149, Val 152, Gly 153, Met 154, Glu 158, Lys 161, Asn 162, Leu 163, Ala167, Lys 168, Thr 171, Gly 196, Val 197, Gly 198, Phe 199, Tyr 200, Val211, Trp 223, Thr 245, Ile 247.

Water molecules, here given the designation Wat1, Wat2, Wat3, Wat9,Wat10, Wat12, Wat13, Wat17, and Wat23, are also part of the bindingpocket and are considered to be key parts of the structure. Thus, anaspect of the present invention is the definition of the protein foldand space (or cavity) occupied by the ligand provided by the crystallineform.

Analysis of the crystalline forms of the present invention suggests thatconformational shifts allow NECA binding. The residues in the followingtwo ranges exhibit an en bloc movement, compared to similar residues inyeast Hsp90, and by their alternate conformation create part of the NECAbinding pocket that discriminates against other substrates such as ADP.Range 1: Gln 79-Asn 96; Range 2: Gly 164-Gly 198.

The C-terminal amino acids also stabilize the GRP94 LBD. Amino acids Val330 through Met 336 form a series of hydrogen bonds with amino acids lie279 through Lys 285 and stabilize the C-terminus of the GRP94 LBD.

V.G. Generation of Easily-Solved Hsp90 Crystals

The present invention discloses a substantially pure GRP94 LBDpolypeptide in crystalline form. In a preferred embodiment, exemplifiedin the Examples, the GRP94 LBD is crystallized with bound ligand.Crystals can be formed from an Hsp90 or GRP94, or Hsp90 or GRP94 LBD,polypeptide that is usually expressed by a cell culture, such as E.coli. Se-Met substitutions can be introduced during the preparation ofthe protein from bacterial cultures and can act as heavy atomsubstitutions in crystals of an Hsp90 or GRP94, or Hsp90 or GRP94 LBD.This method can be advantageous for the phasing of the crystal, which isa crucial, and sometimes limiting, step in solving the three-dimensionalstructure of a crystallized entity. Thus, the need for generating theheavy metal derivatives traditionally employed in crystallography can beeliminated. After the three-dimensional structure of an Hsp90 or GRP94,or Hsp90 or GRP94 LBD, with or without a ligand bound is determined, theresultant three-dimensional structure can be used in computationalmethods to design synthetic ligands for an Hsp90 or GRP94, or Hsp90 orGRP94 LBD, polypeptide. Further activity structure relationships can bedetermined through routine testing, using assays disclosed herein andknown in the art.

VI. Design and Development of Hsp90 Protein Modulators

The knowledge of the structure of Hsp90 proteins, an aspect of thepresent invention, provides a tool for investigating the mechanism ofaction of Hsp90 proteins in a subject. For example, various computermodels, as described herein, can predict the binding of varioussubstrate molecules to Hsp90 proteins. Upon discovering that suchbinding in fact takes place, knowledge of the protein structure thenallows design and synthesis of small molecules that mimic the functionalbinding of the substrate to the Hsp90 proteins. This is the method of“rational” drug design, further described herein.

Use of the isolated and purified Hsp90 protein structures of the presentinvention in rational drug design is thus provided in accordance withthe present invention. Additional rational drug design techniques aredescribed in U.S. Pat. Nos. 5,834,228; 5,872,011; and 6,136,831.

Thus, in addition to the compounds described herein, other stericallysimilar compounds can be formulated to mimic the key structural regionsof a Hsp90 proteins in general, or of GRP94 or HSP90 in particular. Thegeneration of a structural functional equivalent can be achieved by thetechniques of modeling and chemical design known to those of skill inthe art and described herein. It will be understood that all suchsterically similar constructs fall within the scope of the presentinvention. Programs such as but not limited to RASMOL (BiomolecularStructures Group, Glaxo Wellcome Research & Development Stevenage,Hertfordshire, United Kingdom Version 2.6, August 1995, Version 2.6.4,December 1998, Copyright © Roger Sayle 1992-1999) can be used with theatomic structural coordinates from crystals generated by practicing theinvention or used to practice the invention by generatingthree-dimensional models and/or determining the structures involved inligand binding. Computer programs such as those sold under theregistered trademark INSIGHT II® and such as GRASP (Nicholls et al.(1991) Proteins 11:282) allow for further manipulations and the abilityto introduce new structures. In addition, high throughput binding andbiological activity assays can be devised using purified recombinantprotein and assays discussed herein and known to those of skill in theart in order to refine the activity of a designed ligand.

A method of identifying modulators of the activity of an Hsp90 proteinusing rational drug design is thus provided in accordance with thepresent invention. The method comprises designing a potential modulatorfor an Hsp90 protein of the present invention that will formnon-covalent bonds with amino acids in the ligand binding pocket basedupon the crystalline structure of the GRP94 LBD polypeptide;synthesizing the modulator; and determining whether the potentialmodulator modulates the activity of an Hsp90 protein. In a preferredembodiment, the modulator is designed for a GRP94 or HSP90 polypeptide.Modulators can be synthesized using techniques known to those ofordinary skill in the art.

In an alternative embodiment, a method of designing a modulator of aHsp90 protein in accordance with the present invention comprises: (a)selecting a candidate ligand; (b) determining one or more amino acids ofan Hsp90 protein that interact with the ligand using a three-dimensionalmodel of a crystallized GRP94 LBD polypeptide; (c) identifying in abiological assay for Hsp90 protein activity a degree to which the ligandmodulates the activity of the Hsp90 protein; (d) selecting a chemicalmodification of the ligand wherein the interaction between the aminoacids of the Hsp90 protein and the ligand is predicted to be modulatedby the chemical modification; (e) synthesizing the ligand to form amodified ligand; (f) contacting the modified ligand with the Hsp90protein; (g) identifying in a biological assay for Hsp90 protein adegree to which the modified ligand modulates the biological activity ofthe Hsp90 protein; and (h) comparing the biological activity of theHsp90 protein in the presence of modified ligand with the biologicalactivity of the Hsp90 protein in the presence of the unmodified ligand,whereby a modulator of a Hsp90 protein is designed.

In another embodiment, the present invention also provides methods forthe design of modulators that bind at a site other than the ligandbinding pocket, also called an accessory binding site.

In accordance with a preferred embodiment of the present invention, thestructure coordinates of a crystalline GRP94 LBD polypeptide can be usedto design compounds that bind to a Hsp90 protein, more preferably aGRP94 or HSP90 polypeptide, and alter the properties of a Hsp90 proteinby competitive inhibition, non-competitive inhibition, agonism, or othermechanism.

A second design approach is to probe a Hsp90 protein (preferably GRP94or HSP90) crystal with molecules comprising a variety of differentchemical entities to determine optimal sites for interaction betweencandidate Hsp90 protein modulators and the polypeptide. For example,high resolution X-ray diffraction data collected from crystals saturatedwith solvent allows the determination of the site where each type ofsolvent molecule adheres. Small molecules that bind tightly to thosesites can then be designed and synthesized and tested for their capacityto modulate Hsp90 protein activity. Representative approaches aredisclosed in PCT International Publication No. WO 99/26966.

Once a computationally-designed ligand is synthesized using the methodsof the present invention or other methods known to those of skill in theart, assays can be used to establish its efficacy as a modulator ofHsp90 protein (preferably GRP94 or HSP90) activity. The ligands can befurther refined by generating intact Hsp90 protein crystals bound to thesynthetic ligand and determining atomic structural data therefrom. Asdiscussed herein the structure of the ligand can then be furthermodified using methods known to those of skill in the art, in order toimprove the modulation activity or the binding affinity of the ligand.This process can lead to second generation ligands with improvedproperties.

The design of candidate substances, also referred to as “compounds” or“candidate compounds”, that bind to or inhibit activity of an Hsp90protein according to the present invention generally involvesconsideration of two factors. First, the compound must be capable ofphysically and structurally associating with an Hsp90 protein.Non-covalent molecular interactions important in the association of anHsp90 protein with its substrate include hydrogen bonding, van der Waalsinteractions and hydrophobic interactions. Second, the compound must beable to assume a conformation that allows it to associate with an Hsp90protein. Although certain portions of the compound will not directlyparticipate in this association with an Hsp90 protein, those portionscan still influence the overall conformation of the molecule. This, inturn, can have a significant impact on potency. Such conformationalrequirements include the overall three-dimensional structure andorientation of the chemical entity or compound in relation to all or aportion of the binding site, e.g., the ligand binding pocket or anaccessory binding site of a Hsp90 protein, or the spacing betweenfunctional groups of a compound comprising several chemical entitiesthat directly interact with a Hsp90 protein.

The potential modulatory or binding effect of a chemical compound on anHsp90 protein can be analyzed prior to its actual synthesis and testingby the use of computer modeling techniques that employ the coordinatesof a crystalline Hsp90 protein of the present invention. If thetheoretical structure of the given compound suggests insufficientinteraction and association with an Hsp90 protein, synthesis and testingof the compound is obviated. However, if computer modeling indicates astrong interaction, the molecule can then be synthesized and tested forits ability to bind and modulate the activity of an Hsp90 protein. Inthis manner, synthesis of unproductive or inoperative compounds can beminimized.

A modulatory or other binding compound of an Hsp90 protein (preferablyGRP94 or HSP90) can be computationally evaluated and designed via aseries of steps in which chemical entities or fragments are screened andselected for their ability to associate with the individual bindingsites or other areas of a crystalline Hsp90 protein of the presentinvention.

One of several methods can be used to screen chemical entities orfragments for their ability to associate with a Hsp90 protein and, moreparticularly, with the individual binding sites of a GRP94 or HSP90polypeptide, such as ligand binding pocket or an accessory binding site.This process can begin by visual inspection of, for example, the ligandbinding pocket on a computer screen based on the GRP94 LBD atomiccoordinates in Tables 1-2. Selected fragments or chemical entities canthen be positioned in a variety of orientations, or docked, within anindividual binding site of a Hsp90 protein as defined herein above.Docking can be accomplished using software programs such as thoseavailable under the tradenames QUANTA™ (Molecular Simulations Inc., SanDiego, Calif.) and SYBYL™ (Tripos, Inc., St. Louis, Mo.), followed byenergy minimization and molecular dynamics with standard molecularmechanics forcefields, such as CHARM (Brooks et al. (1983) J Comp Chem8:132) and AMBER 5 (Case et al. (1997) AMBER 5, University ofCalifornia, San Francisco, Calif.; Pearlman et al. (1995) Comput PhysCommun 91:1-41).

Specialized computer programs can also assist in the process ofselecting fragments or chemical entities. These include:

1. GRP94ID™ program, version 17 (Goodford (1985) J Med Chem 28:849-857),which is available from Molecular Discovery Ltd., Oxford, UnitedKingdom;

2. MCSS™ program (Miranker & Karplus (1991) Proteins 11:29-34), which isavailable from Molecular Simulations, Inc., San Diego, Calif.;

3. AUTODOCK™ 3.0 program (Goodsell & Olsen (1990) Proteins 8:195-202),which is available from the Scripps Research Institute, La Jolla,Calif.;

4. DOCK™ 4.0 program (Kuntz et al. (1992) J Mol Biol 161:269-288), whichis available from the University of California, San Francisco, Calif.;

5. FLEX-X™ program (See Rarey et al. (1996) J Comput Aid Mol Des10:41-54), which is available from Tripos, Inc., St. Louis, Mo.;

6. MVP program (Lambert (1997) in Practical Application ofComputer-Aided Drug Design, Charifson (ed), pp. 243-303, Marcel-Dekker,New York, N.Y.); and

7. LUDI™ program (Bohm (1992) J Comput Aid Mol Des 6:61-78), which isavailable from Molecular Simulations, Inc., San Diego, Calif.

Once suitable chemical entities or fragments have been selected, theycan be assembled into a single compound or modulator. Assembly canproceed by visual inspection of the relationship of the fragments toeach other on the three-dimensional image displayed on a computer screenin relation to the structure coordinates of a Hsp90 protein. Manualmodel building using software such as QUANTA™ or SYBYL™ typicallyfollows.

Useful programs to aid one of ordinary skill in the art in connectingthe individual chemical entities or fragments include:

1. CAVEAT™ program (Bartlett et al. (1989) Special Pub Royal Chem Soc78: 182-196), which is available from the University of California,Berkeley, Calif.;

2. 3D Database systems, such as MACCS-3D™ system program, which isavailable from MDL Information Systems, San Leandro, Calif. This area isreviewed in Martin (1992) J Med Chem 35:2145-254; and

3. HOOK™ program (Eisen et al. (1994) Proteins 19:199-221), which isavailable from Molecular Simulations, Inc., San Diego, Calif.

Instead of proceeding to build a Hsp90 protein modulator (preferably aGRP94 or HSP90 modulator) in a step-wise fashion one fragment orchemical entity at a time as described above, modulatory or otherbinding compounds can be designed as a whole or de novo using thestructural coordinates of a crystalline Hsp90 protein of the presentinvention and either an empty binding site or optionally including someportion(s) of a known modulator(s). Applicable methods can employ thefollowing software programs:

1. LUDI™ program (Bohm, 1992), which is available from MolecularSimulations, Inc., San Diego, Calif.;

2. LEGEND™ program (Nishibata & Itai (1991) Tetrahedron 47:8985); and

3. LEAPFROG™, which is available from Tripos Associates, St. Louis, Mo.

Other molecular modeling techniques can also be employed in accordancewith this invention. See, e.g., Cohen et al. (1990) J Med Chem33:883-894, Navia & Murcko (1992) Curr Opin Struc Biol 2:202-210, andU.S. Pat. No. 6,008,033.

Once a compound has been designed or selected by the above methods, theefficiency with which that compound can bind to an Hsp90 protein can betested and optimized by computational evaluation. By way of particularexample, a compound that has been designed or selected to function as anHsp90 protein modulator should also preferably traverse a volume notoverlapping that occupied by the binding site when it is bound to aknown ligand. Additionally, an effective Hsp90 protein modulator shouldpreferably demonstrate a relatively small difference in energy betweenits bound and free states (i.e., a small deformation energy of binding).Thus, the most efficient Hsp90 protein modulators should preferably bedesigned with a deformation energy of binding of not greater than about10 kcal/mole, and preferably, not greater than 7 kcal/mole. It ispossible for Hsp90 protein modulators to interact with the polypeptidein more than one conformation that is similar in overall binding energy.In those cases, the deformation energy of binding is taken to be thedifference between the energy of the free compound and the averageenergy of the conformations observed when the modulator binds to thepolypeptide.

A compound designed or selected as binding to an Hsp90 protein(preferably GRP94 or HSP90) can be further computationally optimized sothat in its bound state it would preferably lack repulsive electrostaticinteraction with the target polypeptide. Such non-complementary (e.g.,electrostatic) interactions include repulsive charge-charge,dipole-dipole and charge-dipole interactions. Specifically, the sum ofall electrostatic interactions between the modulator and the polypeptidewhen the modulator is bound to an Hsp90 protein preferably make aneutral or favorable contribution to the enthalpy of binding.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples of programsdesigned for such uses include:

1. Gaussian 98™, which is available from Gaussian, Inc., Pittsburgh,Pa.;

2. AMBER™ program, version 6.0, which is available from the Universityof California at San Francisco;

3. QUANTA™ program, which is available from Molecular Simulations, Inc.,San Diego, Calif.;

4. CHARMm® program, which is available from Molecular Simulations, Inc.,San Diego, Calif.; and

5. Insight II® program, which is available from Molecular Simulations,Inc., San Diego, Calif.

These programs can be implemented using a suitable computer system.Other hardware systems and software packages will be apparent to thoseskilled in the art after review of the disclosure of the presentinvention.

Once an Hsp90 protein modulator has been optimally selected or designed,as described above, substitutions can then be made in some of its atomsor side groups in order to improve or modify its binding properties.Generally, initial substitutions are conservative, i.e., the replacementgroup will have approximately the same size, shape, hydrophobicity andcharge as the original group. It should, of course, be understood thatcomponents known in the art to alter conformation should be avoided.Such substituted chemical compounds can then be analyzed for efficiencyof fit to an Hsp90 protein binding site using the same computer-basedapproaches described in detail above.

VII. Additional Screening Methods Using a Crystalline Hsp90 Protein

The present invention further provides methods for identifyingsubstances that modulate a Hsp90 protein wherein such methods employ acrystalline Hsp90 protein as disclosed herein.

VII.A. Method for Identifying Compounds that Stimulate Hsp90 Activity

In a cell-free system, the method comprises the steps of establishing acontrol system comprising a crystalline Hsp90 protein and a ligand whichis capable of binding to the polypeptide; establishing a test systemcomprising a crystalline Hsp90 protein, the ligand, and a candidatecompound; and determining whether the candidate compound binds thepolypeptide by comparison of the test and control systems. Arepresentative ligand comprises NECA, a substituted adenosine molecule,or a relevant mimetic as obtained through combinatorial chemistry. Thus,in this embodiment, the property screened includes binding affinity.

In another embodiment of the invention, a crystalline form of a Hsp90protein or a catalytic or immunogenic fragment or oligopeptide thereof,can be used for screening libraries of compounds in any of a variety ofdrug screening techniques. The fragment employed in such a screening canbe affixed to a solid support. The formation of binding complexes,between a crystalline Hsp90 protein and the agent being tested, will bedetected. In a preferred embodiment, the crystalline Hsp90 protein is acrystalline GRP94 LBD polypeptide.

Another technique for drug screening which can be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in International Publication No. WO84/03564. According to this method, as applied to a crystallinepolypeptide of the present invention, a multiplicity of different smalltest compounds are synthesized on a solid substrate, such as plasticpins or some other surface. The test compounds are reacted with thecrystalline polypeptide, or fragments thereof. Bound polypeptide is thendetected by methods known to those of skill in the art. The crystallinepolypeptide can also be placed directly onto plates for use in theaforementioned drug screening techniques.

In yet another embodiment, a method of screening for a modulator of aGRP94 or HSP90 polypeptide comprises: providing a library of testsamples; contacting a crystalline form of a GRP94 LBD polypeptide witheach test sample; detecting an interaction between a test sample and acrystalline form of a GRP94 LBD polypeptide; identifying a test samplethat interacts with a crystalline form of a GRP94 LBD polypeptide; andisolating a test sample that interacts with a crystalline form of aGRP94 LBD polypeptide.

In accordance with the present invention there is also provided a rapidand high throughput screening method that relies on the methodsdescribed above. This screening method comprises separately contactingeach of a plurality of samples with a crystalline form of a GRP94 LBDpolypeptide and detecting a resulting binding complex. In such ascreening method, the plurality of samples preferably comprises morethan about 10⁴ samples, or more preferably comprises more than about5×10⁴ samples.

In each of the foregoing embodiments, an interaction can be detectedspectrophotometrically, radiologically, or immunologically. Aninteraction between a crystalline form of a GRP94 LBD polypeptide and atest sample can also be quantified using methodology known to those ofskill in the art. Finally, each of the foregoing embodiments can alsofind application in the screening of non-crystalline forms of an Hsp90polypeptide, such as in the screening of a computationally designedmodulator for the desired effect on the biological activity of the Hsp90polypeptide, including a GRP94 LBD polypeptide of the present invention.Other screening methods pertaining to the biological activity of anHsp90 protein and employing a GRP94 polypeptide are disclosed hereinbelow.

VII.B. Method for Identifying Compounds that Inhibit Hsp90 Activity

The present invention further discloses an assay method for identifyinga compound that inhibits Hsp90 protein transition to or stability of anactive comformation. A ligand of a Hsp90 protein, for example NECA, asubstituted adenosine molecule, related purine nucleoside derivatives, arelevant mimetic as obtained through combinatorial chemistry and/orthose compounds bearing structural similarities to the natural productcompounds geldanamycin and radicicol which bind the GRP94 NBD and andinhibit GRP94 function, can be used in the assay method as the ligandagainst which the inhibition by a test compound is gauged. The methodcomprises (a) incubating a Hsp90 protein with a ligand in the presenceof a test inhibitor compound; (b) determining an amount of ligand thatis bound to the Hsp90 protein, wherein decreased binding of ligand tothe Hsp90 protein in the presence of the test inhibitor compoundrelative to binding in the absence of the test inhibitor compound isindicative of inhibition; and (c) identifying the test compound as aninhibitor of ligand binding if decreased ligand binding is observed.

In another aspect of the present invention, the disclosed assay methodcan be used in the structural refinement of candidate Hsp90 ligands. Forexample, multiple rounds of optimization can be followed by incrementalstructural changes in a strategy of inhibitor design. A strategy such asthis is made possible by the disclosure of the atomic coordinates of theGRP94 LBD polypeptide.

VIII. Design, Preparation, and Structural Analysis of Related Hsp90Proteins

VII.A. Hsp90 Mutants

The present invention provides for the generation of Hsp90 mutants(preferably GRP94 or HSP90 mutants), and the ability to solve thecrystal structures of Hsp90 mutant polypeptides that can becrystallized. More particularly, through the provision of thethree-dimensional structure of GRP94 and HSP90 polypeptides, desirablesites for mutation can be identified.

The structure coordinates of the GRP94 LBD polypeptide provided inaccordance with the present invention also facilitate the identificationand characterization of other proteins that are analogous to Hsp90proteins in function, structure or both. This homology-basedunderstanding can contribute to the development of related therapeuticcompositions and methods.

VIII.B. Hsp90 Protein Equivalents

A further aspect of the present invention is that sterically similarcompounds can be formulated to mimic the key portions of a Hsp90 proteinstructure. Such compounds are functional equivalents. The generation ofa structural functional equivalent can be achieved by the techniques ofmodeling and chemical design known to those of skill in the art anddescribed herein. Modeling and chemical design of Hsp90 proteinstructural equivalents can be based on the structure coordinates of acrystalline GRP94 LBD polypeptide of the present invention. It will beunderstood that all such sterically similar constructs fall within thescope of the present invention.

VIII.C. Solving Crystalline Structures of Related Hsp90 Proteins

Because polypeptides can crystallize in more than one crystal form, thestructural coordinates of a GRP94 LBD polypeptide, or portions thereof,as provided by the present invention, are particularly useful in solvingthe structure of other crystal forms of GRP94, HSP90, and thecrystalline forms of other Hsp90 proteins. The coordinates provided inthe present invention can also be used to solve the structure of Hsp90protein mutants, Hsp90 protein co-complexes, or of the crystalline formof any other protein with significant amino acid sequence homology to afunctional domain of a Hsp90 protein.

One method that can be employed for the purpose of solving additionalHsp90 crystal structures is molecular replacement. See generally,Rossmann, ed, (1972) The Molecular Replacement Method, Gordon & Breach,New York, N.Y. In the molecular replacement method, the unknown crystalstructure, whether it is another crystal form of a GRP94 or HSP90polypeptide, (i.e. a GRP94 or HSP90 mutant), or a GRP94 or HSP90polypeptide complexed with another compound (a “co-complex”), or thecrystal of some other protein with significant amino acid sequencehomology to any functional region of a GRP94 or HSP90 polypeptide, canbe determined using the GRP94 and HSP90 structure coordinates providedin Tables 1-2. This method provides an accurate structural form for theunknown crystal more quickly and efficiently than attempting todetermine such information ab initio.

In addition, in accordance with this invention, GRP94 or HSP90 mutantscan be crystallized in complex with known modulators. The crystalstructures of a series of such complexes can then be solved by molecularreplacement and compared with that of wild type GRP94 or HSP90.Potential sites for modification within the various binding sites of theenzyme can thus be identified. This information provides an additionaltool for determining the most efficient binding interactions, forexample, increased hydrophobic interactions, between a wild type GRP94or HSP90 polypeptide and a ligand when compared to the interactionbetween a mutant GRP94 or HSP90 polypeptide and a ligand.

All of the complexes referred to in the present disclosure can bestudied using X-ray diffraction techniques. See, e.g., Blundell &Johnson (1985) Method Enzymol 114A & 115B and can be refined usingcomputer software, such as the X-PLOR™ program (Brünger, 1992). Thisinformation is thus used to optimize known classes of Hsp90 proteinmodulators, and more importantly, to design and synthesize novel classesof Hsp90 protein modulators.

IX. Design, Preparation and Structural Analysis of GRP94 and GRP94 LBDPolypeptides and Structural Equivalents

The present invention also provides novel purified and isolated Hsp90and GRP94 polypeptides, Hsp90 and GRP94 LBD polypeptides, and mutantsand structural equivalents thereof (preferably GRP94 and GRP94 LBDmutants), and the ability to solve the crystal structures of those thatcrystallize. Thus, an aspect of the present invention involves theproduction of a recombinant protein for, among other things, the purposeof crystallization, characterization of biologically relevantprotein-protein interactions, and compound screening assays, or for theproduction of a recombinant protein having other desirablecharacteristic(s). Polypeptide products produced by the methods of thepresent invention are also disclosed herein.

The structure coordinates of a Hsp90 or GRP94, or Hsp90 or GRP94 LBDprovided in accordance with the present invention also facilitate theidentification of related proteins or enzymes analogous to GRP94 infunction, structure or both, (for example, a GRP94 that can lead tonovel therapeutic modes for treating or preventing a range of diseasestates).

IX.A. GRP94 Nucleic Acids

The nucleic acid molecules provided by the present invention include theisolated nucleic acid molecules of any one of SEQ ID NOs:1, 3, or 5,sequences substantially similar to sequences of any one of SEQ ID NOs:1, 3, or 5, conservative variants thereof, subsequences and elongatedsequences thereof, complementary DNA molecules, and corresponding RNAmolecules. The present invention also encompasses genes, cDNAs, chimericgenes, and vectors comprising disclosed GRP94 nucleic acid sequences. Ina preferred embodiment, a nucleic acid molecule of the present inventionencodes a GRP94 LBD polypeptide. Thus, in a more preferred embodiment, anucleic acid molecule of the present invention is set forth as SEQ IDNO: 3 or 5.

The term “nucleic acid molecule” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides thathave similar properties as the reference natural nucleic acid. Unlessotherwise indicated, a particular nucleotide sequence also implicitlyencompasses conservatively modified variants thereof (e.g. degeneratecodon substitutions), complementary sequences, subsequences, elongatedsequences, as well as the sequence explicitly indicated. The terms“nucleic acid molecule” or “nucleotide sequence” can also be used inplace of “gene”, “cDNA”, or “mRNA”. Nucleic acids can be derived fromany source, including any organism.

The term “isolated”, as used in the context of a nucleic acid molecule,indicates that the nucleic acid molecule exists apart from its nativeenvironment and is not a product of nature. An isolated DNA molecule canexist in a purified form or can exist in a non-native environment suchas a transgenic host cell.

The term “purified”, when applied to a nucleic acid, denotes that thenucleic acid is essentially free of other cellular components with whichit is associated in the natural state. Preferably, a purified nucleicacid molecule is a homogeneous dry or aqueous solution. The term“purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it meansthat the nucleic acid is at least about 50% pure, more preferably atleast about 85% pure, and most preferably at least about 99% pure.

The term “substantially identical”, the context of two nucleotide oramino acid sequences, can also be defined as two or more sequences orsubsequences that have at least 60%, preferably 80%, more preferably90-95%, and most preferably at least 99% nucleotide or amino acidsequence identity, when compared and aligned for maximum correspondence,as measured using one of the following sequence comparison algorithms(described herein below) or by visual inspection. Preferably, thesubstantial identity exists in nucleotide sequences of at least 50residues, more preferably in nucleotide sequence of at least about 100residues, more preferably in nucleotide sequences of at least about 150residues, and most preferably in nucleotide sequences comprisingcomplete coding sequences. In one aspect, polymorphic sequences can besubstantially identical sequences. The term “polymorphic” refers to theoccurrence of two or more genetically determined alternative sequencesor alleles in a population. An allelic difference can be as small as onebase pair.

Another indication that two nucleotide sequences are substantiallyidentical is that the two molecules specifically or substantiallyhybridize to each other under stringent conditions. In the context ofnucleic acid hybridization, two nucleic acid sequences being comparedcan be designated a “probe” and a “target”. A “probe” is a referencenucleic acid molecule, and a “‘target” is a test nucleic acid molecule,often found within a heterogenous population of nucleic acid molecules.A “target sequence” is synonymous with a “test sequence”.

A preferred nucleotide sequence employed for hybridization studies orassays includes probe sequences that are complementary to or mimic atleast an about 14 to 40 nucleotide sequence of a nucleic acid moleculeof the present invention. Preferably, probes comprise 14 to 20nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100,200, 300, or 500 nucleotides or up to the full length of any of thoseset forth as SEQ ID NOs: 1, 3, or 5. Such fragments can be readilyprepared by, for example, directly synthesizing the fragment by chemicalsynthesis, by application of nucleic acid amplification technology, orby introducing selected sequences into recombinant vectors forrecombinant production.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex nucleic acid mixture (e.g., total cellular DNA or RNA). Thephrase “binds substantially to” refers to complementary hybridizationbetween a probe nucleic acid molecule and a target nucleic acid moleculeand embraces minor mismatches that can be accommodated by reducing thestringency of the hybridization media to achieve the desiredhybridization.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern blot analysis are both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, part I chapter2, Elsevier, New York, N.Y. Generally, highly stringent hybridizationand wash conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. Typically, under “stringent conditions” a probewill hybridize specifically to its target subsequence, but to no othersequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor Southern or Northern Blot analysis of complementary nucleic acidshaving more than about 100 complementary residues is overnighthybridization in 50% formamide with 1 mg of heparin at 42° C. An exampleof highly stringent wash conditions is 15 minutes in 0.15 M NaCl at 65°C. An example of stringent wash conditions is 15 minutes in 0.2×SSCbuffer at 65° C. (See Sambrook et al. eds. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. for a description of SSC buffer). Often, a high stringencywash is preceded by a low stringency wash to remove background probesignal. An example of medium stringency wash conditions for a duplex ofmore than about 100 nucleotides, is 15 minutes in 1×SSC at 45° C. Anexample of low stringency wash for a duplex of more than about 100nucleotides, is 15 minutes in 4-6×SSC at 40° C. For short probes (e.g.,about 10 to 50 nucleotides), stringent conditions typically involve saltconcentrations of less than about 1.0 M Na⁺ ion, typically about 0.01 to1.0 M Na⁺ ion concentration (or other salts) at pH 7.0-8.3, and thetemperature is typically at least about 30° C. Stringent conditions canalso be achieved with the addition of destabilizing agents such asformamide. In general, a signal to noise ratio of 2-fold (or higher)than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.

The following are examples of hybridization and wash conditions that canbe used to clone homologous nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the present invention: aprobe nucleotide sequence preferably hybridizes to a target nucleotidesequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. followed by washing in 2×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequencehybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. followed by washing in 0.5×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in0.1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and targetsequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at 65° C.

A further indication that two nucleic acid sequences are substantiallyidentical is that proteins encoded by the nucleic acids aresubstantially identical, share an overall three-dimensional structure,are biologically functional equivalents, or are immunologicallycross-reactive. These terms are defined further under the heading GRP94Polypeptides herein below. Nucleic acid molecules that do not hybridizeto each other under stringent conditions are still substantiallyidentical if the corresponding proteins are substantially identical.This can occur, for example, when two nucleotide sequences aresignificantly degenerate as permitted by the genetic code.

The term “conservatively substituted variants” refers to nucleic acidsequences having degenerate codon substitutions wherein the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcids Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608;Rossolini et al. (1994) Mol Cell Probes 8:91-98).

The term “subsequence” refers to a sequence of nucleic acids thatcomprises a part of a longer nucleic acid sequence. An exemplarysubsequence is a probe, described herein above, or a primer. The term“primer” as used herein refers to a contiguous sequence comprising about8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20nucleotides, and more preferably 20-30 nucleotides of a selected nucleicacid molecule. The primers of the invention encompass oligonucleotidesof sufficient length and appropriate sequence so as to provideinitiation of polymerization on a nucleic acid molecule of the presentinvention.

The term “elongated sequence” refers to an addition of nucleotides (orother analogous molecules) incorporated into the nucleic acid. Forexample, a polymerase (e.g., a DNA polymerase), e.g., a polymerase thatadds sequences at the 3′ terminus of the nucleic acid molecule can beused to provide an elongated sequence. In addition, the nucleotidesequence can be combined with other DNA sequences, such as promoters,promoter regions, enhancers, polyadenylation signals, intronicsequences, additional restriction enzyme sites, multiple cloning sites,and other coding segments.

The term “complementary sequence”, as used herein, indicates twonucleotide sequences that comprise antiparallel nucleotide sequencescapable of pairing with one another upon formation of hydrogen bondsbetween base pairs. As used herein, the term “complementary sequences”means nucleotide sequences which are substantially complementary, as canbe assessed by the same nucleotide comparison set forth above, or isdefined as being capable of hybridizing to the nucleic acid segment inquestion under relatively stringent conditions such as those describedherein. A particular example of a complementary nucleic acid segment isan antisense oligonucleotide.

The term “gene” refers broadly to any segment of DNA associated with abiological function. A gene encompasses sequences including but notlimited to a coding sequence, a promoter region, a cis-regulatorysequence, a non-expressed DNA segment is a specific recognition sequencefor regulatory proteins, a non-expressed DNA segment that contributes togene expression, a DNA segment designed to have desired parameters, orcombinations thereof. A gene can be obtained by a variety of methods,including cloning from a biological sample, synthesis based on known orpredicted sequence information, and recombinant derivation of anexisting sequence.

The term “gene expression” generally refers to the cellular processes bywhich a biologically active polypeptide is produced from a DNA sequence.

The present invention also encompasses chimeric genes comprising thedisclosed GRP94 sequences. The term “chimeric gene”, as used herein,refers to a promoter region operably linked to a GRP94 coding sequence,a nucleotide sequence producing an antisense RNA molecule, a RNAmolecule having tertiary structure, such as a hairpin structure, or adouble-stranded RNA molecule.

The term “operably linked”, as used herein, refers to a promoter regionthat is connected to a nucleotide sequence in such a way that thetranscription of that nucleotide sequence is controlled and regulated bythat promoter region. Techniques for operatively linking a promoterregion to a nucleotide sequence are well known in the art.

The terms “heterologous gene”, “heterologous DNA sequence”,“heterologous nucleotide sequence”, “exogenous nucleic acid molecule”,or “exogenous DNA segment”, as used herein, each refer to a sequencethat originates from a source foreign to an intended host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified, for example bymutagenesis or by isolation from native cis-regulatory sequences. Theterms also include non-naturally occurring multiple copies of anaturally occurring nucleotide sequence. Thus, the terms refer to a DNAsegment that is foreign or heterologous to the cell, or homologous tothe cell but in a position within the host cell nucleic acid wherein theelement is not ordinarily found.

The term “promoter region” defines a nucleotide sequence within a genethat is positioned 5′ to a coding sequence of a same gene and functionsto direct transcription of the coding sequence. The promoter regionincludes a transcriptional start site and at least one cis-regulatoryelement. The present invention encompasses nucleic acid sequences thatcomprise a promoter region of a GRP94 gene, or functional portionthereof.

The term “cis-acting regulatory sequence” or “cis-regulatory motif” or“response element”, as used herein, each refer to a nucleotide sequencethat enables responsiveness to a regulatory transcription factor.Responsiveness can encompass a decrease or an increase intranscriptional output and is mediated by binding of the transcriptionfactor to the DNA molecule comprising the response element.

As used herein, the term “transcription” means a cellular processinvolving the interaction of an RNA polymerase with a gene that directsthe expression as RNA of the structural information present in thecoding sequences of the gene. The process includes, but is not limitedto the following steps: (a) the transcription initiation, (b) transcriptelongation, (c) transcript splicing, (d) transcript capping, (e)transcript termination, (f) transcript polyadenylation, (g) nuclearexport of the transcript, (h) transcript editing, and (i) stabilizingthe transcript.

The term “transcription factor” generally refers to a protein thatmodulates gene expression by interaction with the cis-regulatory elementand cellular components for transcription, including RNA Polymerase,Transcription Associated Factors (TAFs), chromatin-remodeling proteins,and any other relevant protein that impacts gene transcription.

A “functional portion” of a promoter gene fragment is a nucleotidesequence within a promoter region that is required for normal genetranscription. To determine nucleotide sequences that are functional,the expression of a reporter gene is assayed when variably placed underthe direction of a promoter region fragment.

Promoter region fragments can be conveniently made by enzymaticdigestion of a larger fragment using restriction endonucleases or DNAseI. Preferably, a functional promoter region fragment comprises about5000 nucleotides, more preferably 2000 nucleotides, more preferablyabout 1000 nucleotides. Even more preferably a functional promoterregion fragment comprises about 500 nucleotides, even more preferably afunctional promoter region fragment comprises about 100 nucleotides, andeven more preferably a functional promoter region fragment comprisesabout 20 nucleotides.

The terms “reporter gene” or “marker gene” or “selectable marker” eachrefer to a heterologous gene encoding a product that is readily observedand/or quantitated. A reporter gene is heterologous in that itoriginates from a source foreign to an intended host cell or, if fromthe same source, is modified from its original form. Non-limitingexamples of detectable reporter genes that can be operably linked to atranscriptional regulatory region can be found in Alam & Cook (1990)Anal Biochem 188:245-254 and PCT International Publication No. WO97/47763. Preferred reporter genes for transcriptional analyses includethe lacZ gene (See, e.g., Rose & Botstein (1983) Meth Enzymol101:167-180), Green Fluorescent Protein (GFP) (Cubitt et al. (1995)Trends Biochem Sci 20:448-455), luciferase, or chloramphenicol acetyltransferase (CAT). Preferred reporter genes for methods to producetransgenic animals include but are not limited to antibiotic resistancegenes, and more preferably the antibiotic resistance gene confersneomycin resistance. Any suitable reporter and detection method can beused, and it will be appreciated by one of skill in the art that noparticular choice is essential to or a limitation of the presentinvention.

An amount of reporter gene can be assayed by any method forqualitatively or preferably, quantitatively determining presence oractivity of the reporter gene product. The amount of reporter geneexpression directed by each test promoter region fragment is compared toan amount of reporter gene expression to a control construct comprisingthe reporter gene in the absence of a promoter region fragment. Apromoter region fragment is identified as having promoter activity whenthere is significant increase in an amount of reporter gene expressionin a test construct as compared to a control construct. The term“significant increase”, as used herein, refers to an quantified changein a measurable quality that is larger than the margin of error inherentin the measurement technique, preferably an increase by about 2-fold orgreater relative to a control measurement, more preferably an increaseby about 5-fold or greater, and most preferably an increase by about10-fold or greater.

The present invention further includes vectors comprising the disclosedGRP94 sequences, including plasmids, cosmids, and viral vectors. Theterm “vector”, as used herein refers to a DNA molecule having sequencesthat enable its replication in a compatible host cell. A vector alsoincludes nucleotide sequences to permit ligation of nucleotide sequenceswithin the vector, wherein such nucleotide sequences are also replicatedin a compatible host cell. A vector can also mediate recombinantproduction of a GRP94 polypeptide, as described further herein below.Preferred vectors are listed above under the heading Production of Hsp90Polypeptide and also include but are not limited to pBluescript(Stratagene), pUC18, pBLCAT3 (Luckow & Schutz (1987) Nucleic Acids Res15:5490), pLNTK (Gorman et al. (1996) Immunity 5:241-252), and pBAD/gIII(Stratagene). A preferred host cell is a mammalian cell; more preferablythe cell is a Chinese hamster ovary cell, a HeLa cell, a baby hamsterkidney cell, or a mouse cell; even more preferably the cell is a humancell.

Nucleic acids of the present invention can be cloned, synthesized,recombinantly altered, mutagenized, or combinations thereof. Standardrecombinant DNA and molecular cloning techniques used to isolate nucleicacids are well known in the art. Exemplary, non-limiting methods aredescribed by Sambrook et al., eds. (1989); by Silhavy et al. (1984)Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; by Ausubel et al. (1992) Current Protocols inMolecular Biology, John Wylie and Sons, Inc., New York, N.Y.; and byGlover, ed. (1985) DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, United Kingdom. Site-specific mutagenesis to create base pairchanges, deletions, or small insertions are also well known in the artas exemplified by publications, see, e.g., Adelman et al., (1983) DNA2:183; Sambrook et al. (1989).

Sequences detected by methods of the invention can be detected,subcloned, sequenced, and further evaluated by any measure well known inthe art using any method usually applied to the detection of a specificDNA sequence including but not limited to dideoxy sequencing, PCR,oligomer restriction (Saiki et al. (1985) Bio/Technology 3:1008-1012),allele-specific oligonucleotide (ASO) probe analysis (Conner et al.(1983) Proc Natl Acad Sci USA 80:278), and oligonucleotide ligationassays (OLAs) (Landgren et. al. (1988) Science 241:1007). Moleculartechniques for DNA analysis have been reviewed (Landgren et. al. (1988)Science 242:229-237).

IX.B. GRP94 Polypeptides

The polypeptides provided by the present invention include the isolatedpolypeptides set forth as SEQ ID NOs:2, 4 or 6, polypeptidessubstantially identical to SEQ ID NOs:2, 4 or 6, GRP94 polypeptidefragments, fusion proteins comprising GRP94 amino acid sequences,biologically functional analogs, and polypeptides that cross-react withan antibody that specifically recognizes a GRP94 polypeptide. In apreferred embodiment the GRP94 polypeptide is a GRP94 LBD polypeptide.Thus, in a more preferred embodiment, a GRP94LBD comprises the aminoacid sequence of any of SEQ ID NOS:4 or 6.

The term “isolated”, as used in the context of a polypeptide, indicatesthat the polypeptide exists apart from its native environment and is nota product of nature. An isolated polypeptide can exist in a purifiedform or can exist in a non-native environment such as, for example, in atransgenic host cell.

The term “purified”, when applied to a polypeptide, denotes that thepolypeptide is essentially free of other cellular components with whichit is associated in the natural state. Preferably, a polypeptide is ahomogeneous solid or aqueous solution. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A polypeptide that is the predominant species present ina preparation is substantially purified. The term “purified” denotesthat a polypeptide gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the polypeptide is atleast about 50% pure, more preferably at least about 85% pure, and mostpreferably at least about 99% pure.

The term “substantially identical” in the context of two or morepolypeptides sequences is measured by (a) polypeptide sequences havingabout 35%, or 45%, or preferably from 45-55%, or more preferably 55-65%,or most preferably 65% or greater amino acids that are identical orfunctionally equivalent. Percent “identity” and methods for determiningidentity are defined herein below.

Substantially identical polypeptides also encompass two or morepolypeptides sharing a conserved three-dimensional structure.Computational methods can be used to compare structural representations,and structural models can be generated and easily tuned to identifysimilarities around important active sites or ligand binding sites. SeeHenikoff et al. (2000) Electrophoresis 21(9):1700-1706; Huang et al.(2000) Pac Symp Biocomput 230-241; Saqi et al. (1999) Bioinformatics15(6):521-522; and Barton (1998) Acta Crystallogr D Biol Crystallogr54:1139-1146.

The term “functionally equivalent” in the context of amino acidsequences is well known in the art and is based on the relativesimilarity of the amino acid side-chain substituents. See Henikoff &Henikoff (2000) Adv Protein Chem 54:73-97. Relevant factors forconsideration include side-chain hydrophobicity, hydrophilicity, charge,and size. For example, arginine, lysine, and histidine are allpositively charged residues; that alanine, glycine, and serine are allof similar size; and that phenylalanine, tryptophan, and tyrosine allhave a generally similar shape. By this analysis, described furtherherein below, arginine, lysine, and histidine; alanine, glycine, andserine; and phenylalanine, tryptophan, and tyrosine; are defined hereinas biologically functional equivalents.

In making biologically functional equivalent amino acid substitutions,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics, these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al. (1982) J Mol Biol 157:105.). It is known thatcertain amino acids can be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ofthe original value is preferred, those which are within ±1 of theoriginal value are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with its immunogenicity and antigenicity, i.e. with abiological property of the protein. It is understood that an amino acidcan be substituted for another having a similar hydrophilicity value andstill obtain a biologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofthe original value is preferred, those which are within ±1 of theoriginal value are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

The present invention also encompasses GRP94 polypeptide fragments orfunctional portions of a GRP94 polypeptide. Such functional portion neednot comprise all or substantially all of the amino acid sequence of anative GRP94 gene product. The term “functional” includes any biologicalactivity or feature of GRP94, including immunogenicity. Preferredembodiments include a GRP94 LBD and a fragment thereof defining theligand binding pocket.

The present invention also includes longer sequences of a GRP94polypeptide, or portion thereof. For example, one or more amino acidscan be added to the N-terminus or C-terminus of a GRP94 polypeptide.Fusion proteins comprising GRP94 polypeptide sequences are also providedwithin the scope of the present invention. Methods of preparing suchproteins are known in the art.

The present invention also encompasses functional analogs of a GRP94polypeptide. Functional analogs share at least one biological functionwith a GRP94 polypeptide. An exemplary function is immunogenicity. Inthe context of amino acid sequence, biologically functional analogs, asused herein, are peptides in which certain, but not most or all, of theamino acids can be substituted. Functional analogs can be created at thelevel of the corresponding nucleic acid molecule, altering such sequenceto encode desired amino acid changes. In one embodiment, changes can beintroduced to improve the antigenicity of the protein. In anotherembodiment, a GRP94 polypeptide sequence is varied so as to assess theactivity of a mutant GRP94 polypeptide.

The present invention also encompasses recombinant production of thedisclosed GRP94 polypeptides. Briefly, a nucleic acid sequence encodinga GRP94 polypeptide, or portion thereof, is cloned into a expressioncassette, the cassette is introduced into a host organism, where it isrecombinantly produced.

The term “expression cassette” as used herein means a DNA sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassettecomprising the nucleotide sequence of interest can be chimeric. Theexpression cassette can also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.

The expression of the nucleotide sequence in the expression cassette canbe under the control of a constitutive promoter or an induciblepromoter, which initiates transcription only when the host cell isexposed to some particular external stimulus. Exemplary promotersinclude Simian virus 40 early promoter, a long terminal repeat promoterfrom retrovirus, an action promoter, a heat shock promoter, and ametallothien protein. In the case of a multicellular organism, thepromoter and promoter region can direct expression to a particulartissue or organ or stage of development. Exemplary tissue-specificpromoter regions include a GRP94 promoter, described herein. Suitableexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus, yeast vectors, bacteriophage vectors(e.g., lambda phage), and plasmid and cosmids DNA vectors.

The term “host cell”, as used herein, refers to a cell into which aheterologous nucleic acid molecule has been introduced. Transformedcells, tissues, or organisms are understood to encompass not only theend product of a transformation process, but also transgenic progenythereof.

A host cell strain can be chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. For example, different host cells havecharacteristic and specific mechanisms for the translational andpost-transactional processing and modification (e.g., glycosylation,phosphorylation of proteins). Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. Expression in a bacterial system can be usedto produce a non-glycosylated core protein product. Expression in yeastwill produce a glycosylated product. Expression in animal cells can beused to ensure “native” glycosylation of a heterologous protein.

Expression constructs are transfected into a host cell by any standardmethod, including electroporation, calcium phosphate precipitation,DEAE-Dextran transfection, liposome-mediated transfection, and infectionusing a retrovirus. The GRP94-encoding nucleotide sequence carried inthe expression construct can be stably integrated into the genome of thehost or it can be present as an extrachromosomal molecule.

Isolated polypeptides and recombinantly produced polypeptides can bepurified and characterized using a variety of standard techniques thatare well known to the skilled artisan. See e.g. Ausubel et al. (1992),Bodanszky, et al. (1976) Peptide Synthesis, John Wiley and Sons, SecondEdition, New York, N.Y. and Zimmer et al. (1993) Peptides, pp. 393-394,ESCOM Science Publishers, B. V.

IX.C. Nucleotide and Amino Acid Sequence Comparisons

The terms “identical” or percent “identity” in the context of two ormore nucleotide or polypeptide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms disclosed herein or by visual inspection.

The term “substantially identical” in regards to a nucleotide orpolypeptide sequence means that a particular sequence varies from thesequence of a naturally occurring sequence by one or more deletions,substitutions, or additions, the net effect of which is to retain atleast some of biological activity of the natural gene, gene product, orsequence. Such sequences include “mutant” sequences, or sequenceswherein the biological activity is altered to some degree but retains atleast some of the original biological activity. The term “naturallyoccurring”, as used herein, is used to describe a composition that canbe found in nature as distinct from being artificially produced by man.For example, a protein or nucleotide sequence present in an organism,which can be isolated from a source in nature and which has not beenintentionally modified by man in the laboratory, is naturally occurring.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer program, subsequence coordinates are designated if necessary,and sequence algorithm program parameters are selected. The sequencecomparison algorithm then calculates the percent sequence identity forthe designated test sequence(s) relative to the reference sequence,based on the selected program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman (1981) Adv Appl Math2:482, by the homology alignment algorithm of Needleman & Wunsch (1970)J Mol Biol 48:443, by the search for similarity method of Pearson &Lipman (1988) Proc Natl Acad Sci USA 85:2444-2448, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group,Madison, Wis.), or by visual inspection. See generally, Ausubel et al.,1992.

A preferred algorithm for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al. (1990) J Mol Biol 215: 403-410. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer high scoringsequence pairs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength W=11, an expectationE=10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. SeeHenikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. See, e.g., Karlin and Altschul (1993) Proc Natl Acad SciUSA 90:5873-5887. One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

IX.D. Hsp90 and GRP94 Mutant Polypeptides

The generation of chimeric GRP94 polypeptides is also an aspect of thepresent invention. Such a chimeric polypeptide can comprise a GRP94, ora GRP94 LBD polypeptide or a portion of a GRP94 or a GRP94 LBD, (e.g. abinding pocket of a GRP94 LBD) that is fused to a candidate polypeptideor a suitable region of the candidate polypeptide. Throughout thepresent disclosure it is intended that the term “mutant” encompass notonly mutants of a polypeptide but chimeric proteins generated using aGRP94 or a GRP94 LBD, as well. It is thus intended that the followingdiscussion of a mutant GRP94 or GRP94 LBD apply mutatis mutandis tochimeric GRP94 and GRP94 LBD polypeptides and and to structuralequivalents thereof.

In accordance with the present invention, a mutation can be directed toa particular site or combination of sites of a wild-type GRP94 or GRP94LBD polypeptide. For example, an accessory binding site or the bindingpocket can be chosen for mutagenesis. Similarly, a residue having alocation on, at or near the surface of the polypeptide can be replaced,resulting in an altered surface charge of one or more charge units, ascompared to the wild-type GRP94 or GRP94 LBD polypeptide. Alternatively,an amino acid residue in a GRP94 or GRP94 LBD polypeptide can be chosenfor replacement based on its hydrophilic or hydrophobic characteristics.

Such mutants can be characterized by any one of several differentproperties, i.e. a “desired” or “predetermined” characteristic a'scompared with the wild type GRP94 or a GRP94 LBD polypeptide. Forexample, such mutants can have an altered surface charge of one or morecharge units, or can have an increase in overall stability. Othermutants can have altered substrate specificity in comparison with, or ahigher specific activity than, a wild-type GRP94 or a GRP94 LBDpolypeptide.

GRP94 or GRP94 LBD polypeptide mutants of the present invention can begenerated in a number of ways. For example, the wild-type sequence of aGRP94 or a GRP94 LBD polypeptide can be mutated at those sitesidentified using this invention as desirable for mutation, by theapproach of oligonucleotide-directed mutagenesis or other conventionalmethods, such as deletion. Alternatively, mutants of a GRP94 or a GRP94LBD polypeptide can be generated by the site-specific replacement of aparticular amino acid with an unnaturally occurring amino acid. Inaddition, GRP94 or GRP94 LBD polypeptide mutants can be generatedthrough replacement of an amino acid residue, for example, a particularcysteine or methionine residue, with selenocysteine or selenomethionine.This can be achieved by growing a host organism capable of expressingeither the wild-type or mutant polypeptide on a growth medium depletedof either natural cysteine or methionine (or both) but enriched inselenocysteine or selenomethionine (or both).

Mutations can be introduced into a DNA sequence coding for a GRP94 orGRP94 LBD polypeptide using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites. Mutations can be generated in the full-length DNAsequence of a GRP94 or a GRP94 LBD polypeptide or in any sequence codingfor polypeptide fragments of a GRP94 or a GRP94 LBD polypeptide.

According to the present invention, a mutated GRP94 or a GRP94 LBDpolypeptide-encoding DNA sequence produced by the methods describedabove, or any alternative methods known in the art, can be expressedusing an expression vector in accordance with techniques disclosedherein above. Subsequently, the polypeptide can be purified and/orcrystallized in accordance with techniques disclosed herein.

Once a mutation(s) has been generated in the desired location, such asan active site, the mutants can be tested for any one of severalproperties of interest, i.e. “desired” or “predetermined” positions. Forexample, mutants can be screened for an altered charge at physiologicalpH. This property can be determined by measuring the mutant polypeptideisoelectric point (pI) and comparing the observed value with that of thewild-type parent. Isoelectric point can be measured bygel-electrophoresis according to the method of Wellner (Wellner, (1971)Anal. Chem. 43: 597). A mutant polypeptide containing a replacementamino acid located at the surface of the enzyme, as provided by thestructural information of this invention, can lead to an altered surfacecharge and an altered pI.

IX.E. Antibodies to a GRP94 Polypeptide of the Present Invention

The present invention also provides an antibody that specifically bindsa GRP94 or a GRP94 LBD polypeptide and methods to generate same. Theterm “antibody” indicates an immunoglobulin protein, or functionalportion thereof, including a polyclonal antibody, a monoclonal antibody,a chimeric antibody, a single chain antibody, Fab fragments, and a Fabexpression library. “Functional portion” refers to the part of theprotein that binds a molecule of interest. In a preferred embodiment, anantibody of the invention is a monoclonal antibody. Techniques forpreparing and characterizing antibodies are well known in the art (See,e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). A monoclonalantibody of the present invention can be readily prepared through use ofwell-known techniques such as the hybridoma techniques exemplified inU.S. Pat. No. 4,196,265 and the phage-displayed techniques disclosed inU.S. Pat. No. 5,260,203.

The phrase “specifically (or selectively) binds to an antibody”, or“specifically (or selectively) immunoreactive with”, when referring to aprotein or peptide, refers to a binding reaction which is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biological materials. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein and donot show significant binding to other proteins present in the sample.Specific binding to an antibody under such conditions can require anantibody that is selected for its specificity for a particular protein.For example, antibodies raised to a protein with an amino acid sequenceencoded by any of the nucleic acid sequences of the invention can beselected to obtain antibodies specifically immunoreactive with thatprotein and not with unrelated proteins.

The use of a molecular cloning approach to generate antibodies,particularly monoclonal antibodies, and more particularly single chainmonoclonal antibodies, are also provided. The production of single chainantibodies has been described in the art. See, e.g., U.S. Pat. No.5,260,203. For this approach, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning on endothelial tissue. The advantages of thisapproach over conventional hybridoma techniques are that approximately10⁴ times as many antibodies can be produced and screened in a singleround, and that new specificities are generated by heavy (H) and light(L) chain combinations in a single chain, which further increases thechance of finding appropriate antibodies. Thus, an antibody of thepresent invention, or a “derivative” of an antibody of the presentinvention, pertains to a single polypeptide chain binding molecule whichhas binding specificity and affinity substantially similar to thebinding specificity and affinity of the light and heavy chain aggregatevariable region of an antibody described herein.

The term “immunochemical reaction”, as used herein, refers to any of avariety of immunoassay formats used to detect antibodies specificallybound to a particular protein, including but not limited to competitiveand non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. See Harlow & Lane (1988) for a description of immunoassayformats and conditions.

IX.F. Methods for Detecting a GRP94 or a GRP94 LBD Polypeptide or forDetecting a Nucleic Acid Molecule Encoding the Same

In another aspect of the invention, a method is provided for detecting alevel of a GRP94 or a GRP94 LBD polypeptide using an antibody thatspecifically recognizes a GRP94 or a GRP94 LBD polypeptide, or portionthereof. In a preferred embodiment, biological samples from anexperimental subject and a control subject are obtained, and a GRP94 orGRP94 LBD polypeptide is detected in each sample by immunochemicalreaction with the antibody. More preferably, the antibody recognizesamino acids of any one of SEQ ID NOs:2, 4 and 6, and is preparedaccording to a method of the present invention for producing such anantibody.

In one embodiment, an antibody is used to screen a biological sample forthe presence of a GRP94 or a GRP94 LBD polypeptide. A biological sampleto be screened can be a biological fluid such as extracellular orintracellular fluid, or a cell or tissue extract or homogenate. Abiological sample can also be an isolated cell (e.g., in culture) or acollection of cells such as in a tissue sample or histology sample. Atissue sample can be suspended in a liquid medium or fixed onto a solidsupport such as a microscope slide. In accordance with a screening assaymethod, a biological sample is exposed to an antibody immunoreactivewith a GRP94 or a GRP94 LBD polypeptide whose presence is being assayed,and the formation of antibody-polypeptide complexes is detected.Techniques for detecting such antibody-antigen conjugates or complexesare well known in the art and include but are not limited tocentrifugation, affinity chromatography and the like, and binding of alabeled secondary antibody to the antibody-antigen complex.

In another aspect of the invention, a method is provided for detecting anucleic acid molecule that encodes a GRP94 or a GRP94 LBD polypeptide.According to the method, a biological sample having nucleic acidmaterial is procured and hybridized under stringent hybridizationconditions to a GRP94 or a GRP94 LBD polypeptide-encoding nucleic acidmolecule of the present invention. Such hybridization enables a nucleicacid molecule of the biological sample and a GRP94 or a GRP94 LBDpolypeptide-encoding nucleic acid molecule to form a detectable duplexstructure. Preferably, the GRP94 or a GRP94 LBD polypeptide-encodingnucleic acid molecule includes some or all nucleotides of any one of SEQID NOs:3 or 5. Also preferably, the biological sample comprises humannucleic acid material.

X. Ligand Compositions

In one embodiment the present invention pertains to a composition ofmatter that acts as a ligand for GRP94. Such a ligand can be identifiedusing the methods disclosed herein and can also be used in thepreparation of a crystalline structure of the present invention. Theligand can comprise a purified and isolated natural ligand for GRP94, orcan comprise a synthetic compound, such as are identified by thescreening and rational drug design techniques disclosed herein.Preferably, the ligand is a small molecule mimetic. More preferably, theligand has activity in the modulation of GRP94 biological activity.Thus, ligands having such activity are also referred to herein as“modulators”. Representative ligand compositions are preferably about500-1000 daltons, polycyclic molecules that can show structuralresemblance to radicicol, geldanamycin, or adenosine derivatives.Optionally, a ligand is hydrophobic.

A representative ligand or modulator composition of matter comprises anadenosine moiety or structural mimetic thereof having any of a varietyof substitutions at the 2′, 3′, and 5′ positions, in the case ofadenosine, as deemed appropriate by high resolution structural analysesof ligand-GRP94 interactions. Optionally, 5′ position alkyl extensionscan be included, preferably as a carboxamido linkage to the parentadenosine and, to facilitate stable chemical linkage to a solid supportfor the purposes of affinity-based purification, terminating in any of asubset of chemically reactive groups including, but not limited tovinyl, maleimide and/or succinimide esters, or substituents suitable forchemical coupling to solid phase supports, such as amino or sulphydrylgroups. The composition acts as a ligand for GRP94 and has applicationin the purification, screening and therapeutic methods disclosed herein.

Additional ligands can be identified through combinatorial chemistry ofa parent precursor molecule bearing a hydrogen bond mimetic, preferablycorresponding to the ribose of adenosine, and a benzimidazole orstructurally related scaffold, corresponding to the adenine base ofadenosine.

A representative ligand or modulator composition comprises a compound ofthe formula (I):

where:

X and Y are the same or different and X and Y═C, N, O or S; and X and Ycan be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen;

R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₁ to C₆hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ toC₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, C₁ to C₆alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈ aryloxy, primary, secondaryor tertiary C₁ to C₆ alkylamino, primary, secondary or tertiary branchedC₁ to C₆ alkylamino, primary, secondary or tertiary cycloalkylamino,primary, secondary or tertiary C₄ to C₈ arylamino, C₁ to C₆alkylcarboxylic acid, branched C₁ to C₆ alkylcarboxylic acid, C₁ to C₆alkylester, branched C₁ to C₆ alkylester, C₄ to C₈ arylcarboxylic acid,C₄ to C₈ arlyester, C₄ to C₈ aryl substituted C₁ to C₆ alkyl, C₄ to C₁₋₂heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in thering, alkyl-substituted or aryl-substituted C₄ to C₁₋₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R¹ is halo where halois chloro, fluoro, bromo, or iodo;

R²=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₁ to C₆hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ toC₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, C₁ to C₆alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈ aryloxy, primary, secondaryor tertiary C₁ to C₆ alkylamino, primary, secondary or tertiary branchedC₁ to C₆ alkylamino, primary, secondary or tertiary cycloalkylamino,primary, secondary or tertiary C₄ to C₈ arylamino, C₁ to C₆alkylcarboxylic acid, branched C₁ to C₆ alkylcarboxylic acid, C₁ to C₆alkylester, branched C₁ to C₆ alkylester, C₄ to C₈ arylcarboxylic acid,C₄ to C₈ arlyester, C₄ to C₈ aryl substituted C₁ to C₆ alkyl, C₄ to C₁₋₂heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in thering, alkyl-substituted or aryl-substituted C₄ to C₁₋₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R² is halo where halois chloro, fluoro, bromo, or iodo; and

R³=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₁ to C₆hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ toC₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, C₁ to C₆alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈ aryloxy, primary, secondaryor tertiary C₁ to C₆ alkylamino, primary, secondary or tertiary branchedC₁ to C₆ alkylamino, primary, secondary or tertiary cycloalkylamino,primary, secondary or tertiary C₄ to C₈ arylamino, C₁ to C₆alkylcarboxylic acid, branched C₁ to C₆ alkylcarboxylic acid, C₁ to C₆alkylester, branched C₁ to C₆ alkylester, C₄ to C₈ arylcarboxylic acid,C₄ to C₈ arlyester, C₄ to C₈ aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in thering, alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R³ is halo where halois chloro, fluoro, bromo, or iodo.

Where the ligand composition further comprises a compound of the formula(II):

where:

X and Y are the same or different and X and Y═C, N, O or S; and X and Ycan be substituted with hydrogen, hydroxyl, or oxygen, includingdouble-bonded oxygen;

R¹=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₁ to C₆hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ toC₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, C₁ to C₆alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈ aryloxy, primary, secondaryor tertiary C₁ to C₆ alkylamino, primary, secondary or tertiary branchedC₁ to C₆ alkylamino, primary, secondary or tertiary cycloalkylamino,primary, secondary or tertiary C₄ to CB arylamino, C₁ to C₆alkylcarboxylic acid, branched C₁ to C₆ alkylcarboxylic acid, C₁ to C₆alkylester, branched C₁ to C₆ alkylester, C₄ to C₈ arylcarboxylic acid,C₄ to C₈ arlyester, C₄ to C₈ aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in thering, alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R¹ is halo where halois chloro, fluoro, bromo, or iodo;

R²=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₁ to C₆hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ toC₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, C₁ to C₆alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈ aryloxy, primary, secondaryor tertiary C₁ to C₆ alkylamino, primary, secondary or tertiary branchedC₁ to C₆ alkylamino, primary, secondary or tertiary cycloalkylamino,primary, secondary or tertiary C₄ to C₈ arylamino, C₁ to C₆alkylcarboxylic acid, branched C₁ to C₆ alkylcarboxylic acid, C₁ to C₆alkylester, branched C₁ to C₆ alkylester, C₄ to C₈ arylcarboxylic acid,C₄ to C₈ arlyester, C₄ to C₈ aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in thering, alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R² is halo where halois chloro, fluoro, bromo, or iodo;

R³=hydrogen, hydroxyl, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₁ to C₆hydroxyalkyl, branched C₁ to C₆ hydroxyalkyl, C₄ to C₈ cycloalkyl, C₁ toC₆ alkenyl, branched C₁ to C₆ alkenyl, C₄ to C₈ cycloalkenyl, C₄ to C₈aryl, C₄ to C₈ aroyl, C₄ to C₈ aryl-substituted C₁ to C₆ alkyl, C₁ to C₆alkoxy, C₁ to C₆ branched alkoxy, C₄ to C₈ aryloxy, primary, secondaryor tertiary C₁ to C₆ alkylamino, primary, secondary or tertiary branchedC₁ to C₆ alkylamino, primary, secondary or tertiary cycloalkylamino,primary, secondary or tertiary C₄ to C₈ arylamino, C₁ to C₆alkylcarboxylic acid, branched C₁ to C₆ alkylcarboxylic acid, C₁ to C₆alkylester, branched C₁ to C₆ alkylester, C₄ to C₈ arylcarboxylic acid,C₄ to C₈ arlyester, C₄ to C₈ aryl substituted C₁ to C₆ alkyl, C₄ to C₁₂heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in thering, alkyl-substituted or aryl-substituted C₄ to C₁₂ heterocyclic orheteropolycyclic alkyl or aryl with O, N or S in the ring; or hydroxyl-,amino-, or halo-substituted versions thereof; or R³ is halo where halois chloro, fluoro, bromo, or iodo; and

R⁴=C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, C₄ to C₈ cycloalkyl with orwithout O, N or S in the ring, C₁ to C₆ alkenyl, branched C₁ to C₆alkenyl, C₄ to C₈ cycloalkenyl with or without O, N or S in the ring, C₄to C₈ aroyl, C₄ to C₈ aryl, C₄ to C₁₂ heterocyclic or heteropolycyclicalkyl or aryl with O, N or S in the ring, C₄ to C₈ aryl-substituted C₁to C₆ alkyl, alkyl-substituted or aryl-substituted C₄ to C₁₂heterocyclic or heteropolycyclic alkyl or aryl with O, N or S in thering, alkyl-substituted C₄ to C₈ aroyl, or alkyl-substituted C₄ to C₈aryl; or hydroxyl-, amino-, or halo-substituted versions thereof wherehalo is chloro, bromo, fluoro or iodo.

XI. Purification Methods

In accordance with the present invention, a method for purifying acomplex comprising GRP94, or in some instances HSP90, by affinitychromatography is provided. The complex preferably comprises GRP94 boundto an antigenic molecule. More preferably, the complex comprises GRP94non-covalently bound to an antigenic molecule. In one embodiment, themethod comprises contacting a sample comprising a GRP94 complex with abinding agent that preferentially binds GRP94, the binding agentimmobilized to a solid phase support, to immobilize the complex to thesolid phase support; collecting the remaining sample; and eluting theGRP94 complex from the solid phase support to give purified GRP94complex in the eluate. By the phrase “a binding agent thatpreferentially binds GRP94” it is meant an agent that preferentiallybinds GRP94 as compared to other molecular entities, including but notlimited to other heat shock proteins.

The binding agent preferably comprises an adenosine moiety or structuralmimetic thereof having any of a variety of substitutions at the 2′, 3′,and 5′ positions, in the case of adenosine, as deemed appropriate byhigh resolution structural analyses of ligand-GRP94 interactions.Optionally, 5′ position alkyl extensions can be included, preferably asa carboxamido linkage to the parent adenosine and, to facilitate stablechemical linkage to a solid support for the purposes of affinity-basedpurification, terminating in any of a subset of chemically reactivegroups including, but not limited to vinyl, maleimide and/or succinimideesters, or substituents suitable for chemical coupling to solid phasesupports, such as amino or sulphydryl groups. More preferably, thebinding agent is free of ATP or ADP. A representative binding agentcomprises a compound of the formula (I) or a compound of formula (II).Another representative binding agent comprisesN-ethylcarboxamidoadenosine (NECA). Additional ligands can be identifiedthrough combinatorial chemistry of a parent precursor molecule bearing ahydrogen bond mimetic, preferably corresponding to the ribose ofadenosine, and a benzimidazole or structurally related scaffold,corresponding to the adenine base of adenosine.

Optionally, the complex bound to the immobilized binding agent is elutedby washing the solid phase support with a buffer comprising aphysiological salts solution containing appropriate concentrations ofthe parent ligand (i.e., the binding agent) to give complex in theeluate. Hence, a complex further comprising the binding agent or elutingligand is also provided in accordance with the present invention. Theeluting ligand will then be removed from the eluate solution by dialysisin buffers appropriate for GMP production including, but not limited to,physiological salts and volatile salts.

The affinity methods disclosed herein above can be used to isolateGRP94-peptide complexes or GRP94 alone, or in some instances,HSP90-peptide complexes, or the HSP90 protein alone, from any eukaryoticcell. For example, tissues, isolated cells, or immortalized eukaryotecell lines infected with a preselected intracellular pathogen, tumorcells or tumor cell lines can be used. The complex can also be obtainedfrom a vertebrate subject, such as a warm-blooded vertebrate, includingmammals and bird. Optionally, the mammal includes, but is not limitedto, human, mouse, pig, rat, ape, monkey, cat, guinea pig, cow, goat andhorse.

In one embodiment, the complex is “autologous” to the vertebratesubject; that is, the complex is isolated from either from the infectedcells or the cancer cells or precancerous cells of the vertebratesubject (e.g., preferably prepared from infected tissues or tumorbiopsies of a vertebrate subject).

Alternatively, the complex is produced in vitro (e.g., wherein a complexwith an exogenous antigenic molecule is desired). Alternatively, GRP94and/or the antigenic molecule can be isolated from a particularvertebrate subject, or from others, or by recombinant production methodsusing a cloned GRP94 originally derived from a particular vertebratesubject or from others. Exogenous antigens and fragments and derivatives(both peptide and non-peptide) thereof for use in complexing with GRP94(or in some instances HSP90), can be selected from among those known inthe art, as well as those readily identified by standard immunoassaysknow in the art by the ability to bind antibody or MHC molecules(antigenicity) or generate immune response (immunogenicity). Complexesof GRP94 and antigenic molecules can be isolated from cancer orprecancerous tissue of a subject, or from a cancer cell line, or can beproduced in vitro (as is necessary in the embodiment in which anexogenous antigen is used as the antigenic molecule).

XI.A. Isolation of Antigenic/Immunogenic Components

A method for isolating or purifying an antigenic molecule associatedwith a complex comprising GRP94, or in some instances HSP90, is alsoprovided in accordance with the present invention. In one embodiment,the method comprises: contacting a sample comprising a complexcomprising an antigenic molecule and GRP94 with a binding agent thatpreferentially binds GRP94, the binding agent immobilized to a solidphase support, to immobilize the complex to the solid phase support;collecting the remaining sample; eluting the complex from the solidphase support to give purified complex in the eluate; and isolating theantigenic molecule from the eluate.

The binding agent preferably comprises an adenosine moiety or structuralmimetic thereof having any of a variety of substitutions at the 2′, 3′,and 5′ positions, in the case of adenosine, as deemed appropriate byhigh resolution structural analyses of ligand-GRP94 interactions.Optionally, 5′ position alkyl extensions can be included, preferably asa carboxamido linkage to the parent adenosine and, to facilitate stablechemical linkage to a solid support for the purposes of affinity-basedpurification, terminating in any of a subset of chemically reactivegroups including, but not limited to vinyl, maleimide and/or succinimideesters, or substituents suitable for chemical coupling to solid phasesupports, such as amino or sulphydryl groups. More preferably, thebinding agent is free of ATP or ADP. A representative binding agentcomprises a compound of formula (I) or a compound of formula (II).Another representative binding agent comprisesN-ethylcarboxamidoadenosine (NECA). Additional ligands can be identifiedthrough combinatorial chemistry of a parent precursor molecule bearing ahydrogen bond mimetic, preferably corresponding to the ribose ofadenosine, and a benzimidazole or structurally related scaffold,corresponding to the adenine base of adenosine.

Optionally, the complex bound to the immobilized binding agent is elutedby washing the solid phase support with a buffer comprising aphysiological salts solution containing appropriate concentrations ofthe parent ligand (i.e. the binding agent) to give complex in theeluate. Hence, a complex further comprising the binding agent or elutingligand is also provided in accordance with the present invention. Theeluting ligand will then be removed from the eluate solution by dialysisin buffers appropriate for GMP production including, but not limited to,physiological salts and volatile salts.

It has been found that antigenic peptides and/or components can beeluted from GRP94-complexes under low pH conditions. These experimentalconditions can be used to isolate peptides and/or antigenic componentsfrom cells that can contain potentially useful antigenic determinants.Once isolated, the amino acid sequence of each antigenic peptide can bedetermined using conventional amino acid sequencing methodologies. Suchantigenic molecules can then be produced by chemical synthesis orrecombinant methods; purified; and complexed to GRP94, or alternativelyHSP90, in vitro. Additionally, antigenic peptide sequences can beobtained by mass spectrometry using, but not limited to, electrosprayand MALDI-TOF instrumentation, coupled with quadrapole detection andCAD-based sequencing.

XI.B. Elution of Peptides from GRP94-Peptide Complexes

Several methods can be used to elute a peptide from a GRP94-peptidecomplex or from a HSP90-peptide complex. The approaches involveincubating the complex in a low pH buffer and/or in guanidinium/HCl (3-6M), 0.1-1% TFA or acetic acid. Briefly, the complex of interest iscentrifuged through a CENTRICON®10 assembly (Amicon of Beverly, Mass.)to remove any low molecular weight material loosely associated with thecomplex. The large molecular weight fraction can be removed and analyzedby SDS-PAGE while the low molecular weight material is fractionated bycapillary and/or nanoscale HPLC, with a flow rate of 0.5 mL/min, withmonitoring at 210/220 nm.

In the low pH protocol, acetic acid or trifluoroacetic acid (TFA) isadded to the complex to give a final concentration of 10% (vol/vol) andthe mixture incubated at room temperature or other suitable temperature,for 10 minutes (Van Bleek et al. (1990) Nature 348:213-216; Li et al.(1993) EMBO J 12:3143-3151).

The resulting samples are centrifuged through a CENTRICON®10 assembly asmentioned previously. The high and low molecular weight fractions arerecovered. The remaining large molecular weight complexes can bereincubated with guanidinium or low pH to remove any remaining peptides.The resulting lower molecular weight fractions are pooled, concentratedby evaporation and dissolved in 0.1% trifluoroacetic acid (TFA). Thedissolved material is fractionated by microbore HPLC, with a flow rateof 0.5 ml/min. The elution of the peptides can be monitored by OD210/220nm and the fractions containing the peptides collected.

XI.C. Sequencing and Synthesis of Peptides

The amino acid sequences of the eluted peptides can be determined eitherby manual or automated amino acid sequencing techniques well known inthe art. Once the amino acid sequence of a potentially protectivepeptide has been determined the peptide can be synthesized in anydesired amount using conventional peptide synthesis or other protocolswell known in the art.

A subject peptide can be synthesized by any of the techniques that areknown to those skilled in the polypeptide art, including recombinant DNAtechniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. Many techniques for peptide synthesis areavailable and can be found in Steward et al. (1969) Solid Phase PeptideSynthesis, W. H. Freeman Co., San Francisco, Calif.; Bodanszky, et al.(1976) Peptide Synthesis, John Wiley & Sons, Second Edition; Meienhofer(1983) Hormonal Proteins and Peptides, Vol. 2, p. 46, Academic Press,New York, N.Y.; Merrifield (1969) Adv Enzymol 32:221-296; Fields et al.(1990) Int J Peptide Protein Res 35:161-214; and U.S. Pat. No. 4,244,946for solid phase peptide synthesis; and Schroder et al. (1965) ThePeptides, Vol. 1, Academic Press, New York, N.Y. for classical solutionsynthesis, each of which is incorporated herein by reference.Appropriate protective groups usable in such synthesis are described inthe above texts and in McOmie (1973) Protective Groups in OrganicChemistry, Plenum Press, New York, N.Y., which is incorporated herein byreference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the final linearpolypeptide.

The resultant linear polypeptides prepared for example as describedabove can be reacted to form their corresponding cyclic peptides. Anexemplary method for cyclizing peptides is described by Zimmer et al.(1993) Peptides, pp. 393-394, ESCOM Science Publishers, B. V. Typically,tertbutoxycarbonyl protected peptide methyl ester is dissolved inmethanol and sodium hydroxide solution are added and the admixture isreacted at 20° C. to hydrolytically remove the methyl ester protectinggroup. After evaporating the solvent, the tertbutoxycarbonyl protectedpeptide is extracted with ethyl acetate from acidified aqueous solvent.The tertbutoxycarbonyl protecting group is then removed under mildlyacidic conditions in dioxane cosolvent. The unprotected linear peptidewith free amino and carboxy termini so obtained is converted to itscorresponding cyclic peptide by reacting a dilute solution of the linearpeptide, in a mixture of dichloromethane and dimethylformamide, withdicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole andN-methylmorpholine. The resultant cyclic peptide is then purified bychromatography.

Purification of the resulting peptides is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

XI.D. Detection Methods

A method for detecting a complex comprising GRP94, or in some instancesHSP90, in a sample suspected of containing such a complex is alsoprovided in accordance with the present invention. In one embodiment,the method comprises: contacting the sample with a binding substancethat preferentially binds GRP94 under conditions favorable to binding acomplex comprising GRP94 to the binding substance to form a secondcomplex there between; and detecting the second complex via a labelconjugated to the binding substance or via a labeled reagent thatspecifically binds to the second complex subsequent to its formation.

The binding substance preferably comprises an adenosine moiety orstructural mimetic thereof having any of a variety of substitutions atthe 2′, 3′, and 5′ positions, in the case of adenosine, as deemedappropriate by high resolution structural analyses of ligand-GRP94interactions. Optionally, 5′ position alkyl extensions can be included,preferably as a carboxamido linkage to the parent adenosine and, tofacilitate stable chemical linkage to a solid support for the purposesof affinity-based purification, terminating in any of a subset ofchemically reactive groups including, but not limited to vinyl,maleimide and/or succinimide esters, or substituents suitable forchemical coupling to solid phase supports, such as amino or sulphydrylgroups. More preferably, the binding substance is free of ATP or ADP. Arepresentative binding substance comprises a compound of formula (I) ora compound of formula (II). Another representative binding substancecomprises N-ethylcarboxamidoadenosine (NECA). Additional ligands can beidentified through combinatorial chemistry of a parent precursormolecule bearing a hydrogen bond mimetic, preferably corresponding tothe ribose of adenosine, and a benzimidazole or structurally relatedscaffold, corresponding to the adenine base of adenosine.

Optionally, the complex bound to the immobilized binding agent is elutedby washing the solid phase support with a buffer comprising aphysiological salts solution containing appropriate concentrations ofthe parent ligand (i.e. the binding substance or agent) to give complexin the eluate. Hence, a complex further comprising the binding agent oreluting ligand is also provided in accordance with the presentinvention. The eluting ligand will then be removed from the eluatesolution by dialysis in buffers appropriate for GMP productionincluding, but not limited to, physiological salts and volatile salts.

The binding substance can be conjugated with a detectable label and inthis case, the detecting step comprises: separating the complex fromunbound labeled binding substance; and detecting the detectable labelwhich is present in the complex or which is unbound.

XI.E. Kits for Purification or Detection

In another aspect, the present invention pertains to a kit for isolatingor purifying a peptide complex, preferably a GRP94 complex, and anantigenic molecule. In one embodiment, the kit comprises a binding agentthat preferentially binds GRP94, the binding agent contained in a firstcontainer. The binding agent preferably comprises an adenosine moiety orstructural mimetic thereof having any of a variety of substitutions atthe 2′, 3′, and 5′ positions, in the case of adenosine, as deemedappropriate by high resolution structural analyses of ligand-GRP94interactions. Optionally, 5′ position alkyl extensions can be included,preferably as a carboxamido linkage to the parent adenosine and, tofacilitate stable chemical linkage to a solid support for the purposesof affinity-based purification, terminating in any of a subset ofchemically reactive groups including, but not limited to vinyl,maleimide and/or succinimide esters, or substituents suitable forchemical coupling to solid phase supports, such as amino or sulphydrylgroups. More preferably, the binding agent is free of ATP or ADP.

A representative binding agent comprises a compound of formula (I) or acompound of formula (II). Another representative binding agent comprisesN-ethylcarboxamidoadenosine (NECA). Additional ligands can be identifiedthrough combinatorial chemistry of a parent precursor molecule bearing ahydrogen bond mimetic, preferably corresponding to the ribose ofadenosine, and a benzimidazole or structurally related scaffold,corresponding to the adenine base of adenosine. Optionally, the bindingagent can be immobilized to a solid phase support, or the kit can alsocomprise a solid phase support contained in a second container.

The kit can further comprise an elution buffer for use in eluting acomplex from the binding agent, the elution buffer contained in a thirdcontainer. Optionally, the elution buffer comprises a physiologicalsalts solution containing appropriate concentrations of the parentligand to give complex in the eluate. The kit can further comprisedialysis buffers appropriate for GMP production including, but notlimited to, physiological salts and volatile salts. The kit can alsofurther comprise an elution buffer for use in eluting an antigenicmolecule from a complex, the elution buffer contained in a fourthcontainer. Suitable elution buffers are disclosed herein above.

In the case of a kit used for detecting a complex comprising GRP94, oralternatively a complex comprising the kit can further comprise areagent or indicator that comprises a detectable label, the indicatorcontaining in a fifth container. Alternatively, the binding agent cancomprise a detectable label or indicator. The indicator can comprise aradioactive label or an enzyme, or other indicator as disclosed herein.

XI.F. Determination of Immunogenicity of GRP94-Peptide Complexes

Purified GRP94-antigenic molecule complexes can be assayed forimmunogenicity using the mixed lymphocyte tumor culture assay (MLTC)well known in the art. By way of example but not limitation, thefollowing procedure can be used. Briefly, mice are injectedsubcutaneously with the candidate GRP94-antigenic molecule complexes.Other mice are injected with either other GRP94-antigenic moleculecomplexes or whole infected cells which act as positive controls for theassay. The mice are injected twice, 7-10 days apart. Ten days after thelast immunization, the spleens are removed and the lymphocytes released.The released lymphocytes can be re-stimulated subsequently in vitro bythe addition of dead cells that expressed the complex of interest.

For example, 8×10⁶ immune spleen cells can be stimulated with 4×10⁴mitomycin C treated or γ-irradiated (5-10,000 rads) infected cells (orcells transfected with an appropriate gene, as the case can be) in 3 mlRPMI medium containing 10% fetal calf serum. In certain cases 33%secondary mixed lymphocyte culture supernatant can be included in theculture medium as a source of T cell growth factors, such as isdescribed by Glasebrook et al. (1980) J Exp Med 151:876. To test theprimary cytotoxic T cell response after immunization, spleen cells canbe cultured without stimulation. In some experiments spleen cells of theimmunized mice can also be re-stimulated with antigenically distinctcells, to determine the specificity of the cytotoxic T cell response.

Six days later the cultures are tested for cytotoxicity in a 4 hour⁵¹Cr-release assay as is described by Palladino et al. (1987) Cancer Res47:5074-5079 and Blachere et al. (1993) J Immunotherapy 14:352-356. Inthis assay, the mixed lymphocyte culture is added to a target cellsuspension to give different effector:target (E:T) ratios (usually 1:1to 40:1). The target cells are prelabeled by incubating 1×10⁶ targetcells in culture medium containing 200 mCi ⁵¹Cr/ml for one hour at 37°C. The cells are washed three times following labeling. Each assay point(E:T ratio) is performed in triplicate and the appropriate controlsincorporated to measure spontaneous ⁵¹Cr release (no lymphocytes addedto assay) and 100% release (cells lysed with detergent). Afterincubating the cell mixtures for 4 hours, the cells are pelleted bycentrifugation at 200 g for 5 minutes. The amount of ⁵¹Cr released intothe supernatant is measured by a gamma counter. The percent cytotoxicityis measured as cpm in the test sample minus spontaneously released cpmdivided by the total detergent released cpm minus spontaneously releasedcpm.

In order to block the MHC class I cascade a concentrated hybridomasupernatant derived from K-44 hybridoma cells (an anti-MHC class Ihybridoma) is added to the test samples to a final concentration of12.5%.

XII. Screening Methods

Disclosed herein is the molecular basis, as well as a high throughputscreen, for chemical compounds that elicit or inhibit conformationalchanges in the molecular chaperone GRP94, or in some instances HSP90,thereby regulating the chaperone and peptide binding activities of theseproteins.

Also disclosed herein are several new and unique aspects of theregulation of GRP94 structure and function that can be readily exploitedfor purposes of identifying agonists and antagonists (“modulators”) ofGRP94 function. GRP94 expression is upregulated by cellular stressessuch as nutrient deprivation, oxidative stress, heavy metal posioning,hypoxia/anoxia, and other conditions related to ischemia. However, untilthe disclosure of the present invention, the molecular mechanismunderlying this activity remained unknown. Thus, disclosed herein is afunctional correlation to heat shock in the observation that heat shockstimulates the peptide binding and chaperone activity of GRP94. The heatshock response of GRP94, which is responsible for its increased peptidebinding and chaperone activity, is a result of a change in theconformational state of the protein from a closed form to an open,active form.

The heat shock induced conformational change can be blocked by theantitumor drugs geldanamycin and radicicol, thus providing a mechanismof their antitumor activity, namely that geldanamycin and radicicolblock GRP94 conformational transitions, and hence chaperone activity.The functional consequence of such inhibition is that oncogenicsignaling proteins, such as growth factor receptor kinases are notprocessed properly and thus, the cell does not receive the proliferativesignals necessary for transformation. Thus, a chemical compound thatmodulates the conformation of GRP94 can be used to treat a diseasestate, such as cancer, wherein a therapeutic benefit can be provided byinhibiting or blocking the egress of proteins (e.g., growth factors)from the endoplasmic reticulum.

The present invention provides the theoretical and structural basis forthe identification of low molecular weight molecules that bind to arecently crystallized conserved N-terminal domain of HSP90, whichpreviously was identified as the binding site for the anti-tumor druggeldanamycin, and elicit a conformation change that yields a dramaticand substantial increase in (poly)peptide binding activity of GRP94, andin some cases, HSP90. In an alternative embodiment, the identifiedmolecules inhibit conformational activation of GRP94, and in some casesHSP90, similar to the observed modulation of GRP94 and HSP90 bygeldanamycin and/or radicicol.

The present invention is markedly distinguished from current perceptionin the art as to the mechanism of regulation of GRP94 and HSP90function. In current views, the Hsp90 family of molecular chaperones arethought to be regulated by cycles of ATP binding and hydrolysis(Prodromou et al. (1997) Cell 90:65-75). This view of Hsp90 function isbased on the observations that the highly conserved N-terminal domain ofthe protein contains a binding site for ATP and ADP and that X-raycrystallographic structures of the domain in complex with ATP and/or ADPcan be obtained.

In accordance with the present invention, data are provideddemonstrating that the related and relevant domain of the HSP90 paralogGRP94 does not display a specific structural preference for ATP or ADP.In a series of function-directed studies, applicants have furtherdetermined that ATP, ADP, geldanamycin and radicicol block or inhibitthe ability of GRP94 to assume a conformation necessary for chaperoneactivity and/or peptide binding. Thus, ATP and ADP, rather than beingphysiological ligands agonising the activity of GRP94, act as inhibitoryagents for this chaperone.

The identified conformational change in GRP94 is a component of theregulatory cycle of GRP94, as demonstrated in the Examples whereinbis-ANS, which bears structural similarities to adenosine nucleotides,was demonstrated to elicit a tertiary conformational change in GRP94that was accompanied by an activation of molecular chaperone and peptidebinding activity.

Structure of bis-ANS

2K⁺

In accordance with the present invention, also disclosed herein are theprimary structural determinants that define low molecular weightcompounds that bind to the conserved N-terminal domain of GRP94 andeither A) elicit a conformational change in GRP94 that is accompanied byan activation of either peptide binding and/or molecular chaperoneactivity, or B) block or inhibit the ability of GRP94 to access oracquire the described conformation. In the present invention, and aswould be apparent to one of ordinary skill in the art of the regulationof protein structure/function after reviewing the disclosure presentedherein, cells and tissues originating from higher eukaryotes contain anative ligand compound bearing structural similarities to adenosine, yetmay bear substituents at the 2′ and 5′ positions, but lack substituentsat the N6 adenine.

Thus, a native ligand, as well an embodiment of a mimetic thereof, bearsan adenosine moiety or moieties and the adenosine moiety(s) function inthe binding of the ligand to the conserved N-terminal domain of GRP94previously identified as an ATP/ADP binding pocket. Representativeligand compositions are disclosed herein above as formulas (I) and (II).Additional ligands can be identified through combinatorial chemistry ofa parent precursor molecule bearing a hydrogen bond mimetic, preferablycorresponding to the ribose of adenosine, and a benzimidazole orstructurally related scaffold, corresponding to the adenine base ofadenosine. Additional ligands can also be identified via the methoddisclosed herein ablve that employ a three-dimensional structure of theGRP94 LBD.

The binding of a ligand elicits the conformational change that isaccompanied by an activation of chaperone and peptide binding activity.Furthermore, synthesis of the native ligand is likely stimulated byconditions that elicit a disruption in the efficiency of protein foldingand assembly in the ER. These conditions include, but are not limitedto, heat shock, oxidative stress, nutrient deprivation, disruptions inoligosaccharide synthesis and covalent assembly on to nascentglycoproteins, and the presence of excessive levels of heavy metals.

Coincident with the discovery of the functional role for GRP94structural transitions in determining the chaperone activity and themechanism of geldanamycin and radicicol action, a simple and rapidmethod for assaying the conformational state of GRP94 (or alternatively,HSP90) is disclosed herein. A preferred embodiment of this method isbased on the preferential binding of the small synthetic fluorescentprobe, bis-ANS, to the open, or active, conformation of GRP94. bis-ANSbinding yields a dramatic increase in probe fluorescence intensity.bis-ANS is identified herein as a highly sensitive indicator of the heatshock induced conformational change of GRP94. Furthermore, bis-ANSitself can elicit the conformational change in GRP94 necessary for theactivation of peptide binding and chaperone function. Thus, bis-ANS isboth an agonist for GRP94 activation as well as an indicator for therelative state of activation. bis-ANS induces these changes on a slowtime scale, thereby enabling it to be used both as an inducer for a heatshock-like conformational change as well as a probe for conformationalchanges induced by other compounds. Conversely, and as disclosed in theExamples, bis-ANS can be used to identify compounds that block the heatshock-induced conformational changes. Indeed, the screening system ofthe present invention showed that radicicol and geldanamycin, twoanti-tumor agents known to act through GRP94/HSP90, block the conversionof these proteins to the conformation necessary for function.

Another preferred embodiment of this method employs a related syntheticfluorescent probe, 8-ANS. 8-ANS also displays preferential binding tothe active conformation of GRP94. However, unlike bis-ANS, 8-ANSfunctions solely as an indicator and lacks agonist activity. 8-ANS isalso useful in screening assays for discovery of GRP94 modulators.

Therefore, in accordance with the present invention, a method ofscreening candidate compounds for an ability to modulate the biologicalactivity is provided. The screening methods are also used to identify anative or endogenous ligand or ligands for GRP94.

In one embodiment, a candidate substance is a substance whichpotentially can modulate the biological activity of GRP94 by binding orother intermolecular interaction with GRP94. By “modulate” is intendedan increase, decrease, or other alteration of any or all biologicalactivities or properties of GRP94. Thus, a native or endogenous ligandor ligands of GRP94 is also a “candidate substance”. A biological samplesuspected of containing a native or endogenous ligand or ligands is alsoa “candidate substance”. Small molecules and combinatorial libraries ofsmall molecules are also candidate “substances”. A candidate substanceidentified according to a screening assay described herein has theability to modulate GRP94 biological activity. Such a candidatesubstance has utility in the treatment of disorders and conditionswherein modulation of the biological activity of GRP94 is desirable, aswell as in the purification and screening methods disclosed herein.

The present invention thus pertains to the molecular basis for as wellas a high throughput screen for chemical compounds that elicit orinhibit conformational changes in the molecular chaperone GRP94, or insome instances HSP90, thereby regulating the chaperone and peptidebinding activities of these proteins.

XII.A. General Screening Methods

A method of screening candidate substances for an ability to modulateGRP94 and/or HSP90 biological activity is thus provided in accordancewith the present invention. In one embodiment, the method comprises (a)establishing a test sample comprising GRP94 and a ligand for GRP94; (b)administering a candidate substance or a sample suspected of containinga candidate substance to the test sample; and (c) measuring an effect onbinding of GRP94 and the ligand for GRP94 in the test sample to therebydetermine the ability of the candidate substance to modulate GRP94biological activity. Preferably, the GRP94 is a GRP94 LBD polypeptide ofthe present invention as disclosed herein above.

The test sample can further comprise an indicator. The term “indicator”is meant to refer to a chemical species or compound that is readilydetectable using a standard detection technique, such as dark versuslight detection, fluorescence or chemiluminescence spectrophotometry,scintillation spectroscopy, chromatography, liquid chromatography/massspectroscopy (LC/MS), colorimetry, and the like. Representativeindicator compounds thus include, but are not limited to, fluorogenic orfluorescent compounds, chemiluminescent compounds, colorimetriccompounds, UV/VIS absorbing compounds, radionucleotides and combinationsthereof. In a preferred embodiment, the ligand further comprises anindicator. In a more preferred embodiment, the ligand/indicatorcomprises 1,8-anilinonapthalenesulfonate (8-ANS).

The ability of the candidate substance to modulate GRP94 and/or HSP90biological activity can determined in any suitable manner. For example,the ability of the candidate substance to modulate GRP94 and/or HSP90biological activity can determined by: (i) detecting a signal producedby the indicator upon an effect of the candidate substance on binding ofGRP94 and/or HSP90 and the ligand for GRP94 and/or HSP90; and (ii)identifying the candidate substance as a modulator of GRP94 and/or HSP90biological activity based upon an amount of signal produced as comparedto a control sample.

In a preferred embodiment, a simple and effective fluorescence basedscreening methodology is provided to identify inhibitors and activatorsof the conformational transitions of GRP94 that are responsible for itsactivity. The method is readily amenable to both robotic and very highthroughput systems.

Thus, in one embodiment, a screening method of the present inventionpertains to a method for a identifying a candidate substance as anactivator of the biological activity of an Hsp90 protein. In a preferredembodiment, the Hsp90 protein is GRP94 or HSP90. The method comprisesestablishing a test sample comprising an Hsp90 protein and a candidatesubstance; administering 8-ANS to the test sample; and detecting afluorescence signal produced by the 8-ANS; and identifying the candidatesubstance as an activator of the biological activity of the Hsp90protein based upon an amount of fluorescence signal produced by the8-ANS as compared to a control sample.

The method can further comprise incubating the Hsp90 protein with thecandidate substance at 37° C. for about one hour prior to adding the8-ANS. Optionally, the 8-ANS can be added in an approximately equimolaramount to the Hsp90 protein. Additionally, the candidate substance isidentified as an activator of the biological activity of an Hsp90protein by detection of an increased 8-ANS fluorescence signal ascompared to a control sample.

In another embodiment, a screening method of the present inventionpertains to a method for a identifying a candidate substance as aninhibitor of the biological activity of a Hsp90 protein. The methodcomprises establishing a test sample comprising an Hsp90 protein and, acandidate substance; heat-shocking the test sample to induce aconformational change to the Hsp90 protein; administering 8-ANS to thetest sample; detecting a fluorescence signal produced by the 8-ANS; andidentifying the candidate substance as an inhibitor of the biologicalactivity of an Hsp90 protein based upon an amount of fluorescence signalproduced by the 8-ANS as compared to a control sample. In a preferredembodiment, the Hsp90 protein is GRP94 or HSP90.

Optionally, the method can further comprise incubating the test sampleat 37° C. for about one hour prior to heat-shocking the test sample. Theheat-shocking can be carried out at 50° C. for about 15 minutes.Preferably, the 8-ANS is added in an approximately equimolar amount tothe Hsp90 protein. The candidate substance can also be identified as aninhibitor of the biological activity of an Hsp90 protein by detection ofa decreased 8-ANS fluorescence signal as compared to a control sample.

XII.B. Cell Based Screening Assays

A screening assay of the present invention may also involve determiningthe ability of a candidate substance to modulate, i.e. inhibit orpromote the biological activity of an Hsp90 protein such as GRP94 andpreferably, to thereby modulate the biological activity of an Hsp90protein such as GRP94 in target cells. Target cells can be eithernaturally occurring cells known to contain a polypeptide of the presentinvention or transformed cells produced in accordance with a process oftransformation set forth herein above. The test samples can furthercomprise a cell or cell line that expresses an Hsp90 polypeptide; thepresent invention also contemplates a recombinant cell line suitable foruse in the exemplary method. Such cell lines may be mammalian, or human,or they may from another organism, including but not limited to yeast.Preferably, the cells express a GRP94 LBD polypeptide of the presentinvention as disclosed herein above.

Representative assays including genetic screening assays and molecularbiology screens such as a yeast two-hybrid screen that will effectivelyidentify Hsp90-interacting genes important for Hsp90 or otherHsp90-mediated cellular process, including a native Hsp90 ligand orligands. One version of the yeast two-hybrid system has been described(Chien et al. (1991) Proc Natl Acad Sci USA 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.). Thus, inaccordance with one embodiment of a screening assay of the presentinvention, the candidate substance is further characterized as acandidate polypeptide, and the screening method can further comprise thestep of purifying and isolating a nucleic acid molecule encoding thecandidate polypeptide.

Thus, enzymes in the cells of higher eukaryotes that mediate the steadystate and stress-elicited production of a GRP94 and/or HSP90 ligand canalso be modulated in accordance with the present invention. Suchcatabolic enzymes also represent appropriate and rational targets forthe design of compounds that elicit an increase in the steady statelevels of a native Hsp90 ligand (e.g., a native GRP94 or HSP90 ligand)and thereby lead to the elicitation of the structural and functionalactivation of chaperone and peptide binding activity of an Hsp90protein, preferably GRP94, disclosed herein.

A screening assay can provide a cell under conditions suitable fortesting the modulation of biological activity of an Hsp90 protein suchas GRP94. These conditions include but are not limited to pH,temperature, tonicity, the presence of relevant metabolic factors (e.g.,metal ions such as for example Ca⁺⁺, growth factor, interleukins, orcolony stimulating factors), and relevant modifications to thepolypeptide such as glycosylation or prenylation. A polypeptide of thepresent invention can be expressed and utilized in a prokaryotic oreukaryotic cell. The host cell can also be fractionated intosub-cellular fractions where the receptor can be found. For example,cells expressing the polypeptide can be fractionated into the nuclei,the endoplasmic reticulum, vesicles, or the membrane surfaces of thecell. U.S. Pat. Nos. 5,837,479; 5,645,999; 5,786,152; 5,739,278; and5,352,660 also describe exemplary screening assays, and the entirecontents of each are herein incorporated by reference.

XII.C. High Throughput Screening

In another embodiment of the screening method of the present invention,an Hsp90 polypeptide (e.g., human GRP94) or active fragment oroligopeptide thereof (e.g., GRP94 LBD disclosed herein), can be used forscreening libraries of compounds in any of a variety of high throughputdrug screening techniques. The fragment employed in such screening canbe free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The formation of binding complexes,between the Hsp90 polypeptide, preferably a GRP94 polypeptide, morepreferably a GRP94 LBD polypeptide, and the candidate substance beingtested, can be measured as described herein.

XIII. Modulation of Hsp90 Biological Activity

Because Hsp90 proteins are found in essentially every cell of the humanbody and are involved in'the processing of many different cellularproteins as well as the presentation of tumor and foreign antigens tothe immune system, compounds identified through the screening method ofthe present invention and disclosed herein (referred to as “ligandcompositions” or “modulators”) have wide ranging value as therapeuticsand in vaccine development. Representative ligand compositions ormodulators are described herein above as formula (I). Modulators that donot structurally resemble adenosine are also provided, and include thosedesigned and/or identified by the rational drug design and combinatorialscreening methods disclosed hereinabove.

In a preferred embodiment, the Hsp90 modulator elicits a conformationalchange in an Hsp90 protein. Even more preferably, the Hsp90 proteinactivity modulator is identified according to a screening assaydescribed herein. A modulator can modulate the biological activity of anHsp90 protein such as GRP94. Relevant to the antigen-presenting activityof GRP94 and HSP90, activators thereof can be applied in vitro to assistin peptide loading onto these proteins for the production of vaccinesdirected against the tissues or invasive organisms possessing thosespecific peptide epitopes. Activators of GRP94/HSP90 biological activitycan be applied to tumor cells excised from cancer patients to increasethe antigenicity of the tumor cells prior to lethal inactivation of thecells and their re-injection into the body as immunostimulatory agents.Activators of GRP94/HSP90 biological activity can be administereddirectly into the body of a vertebrate for increasing the antigenicityof tumors in situ. Activators of GRP94/HSP90 biological activity canalso have antibiotic action against bacteria, viruses, or internalparasites by increasing the antigenicity of the bacteria, virus, orparasites and recognition of same by the adaptive immune system.Activators of GRP94/HSP90 biological activity can be used in furtherscreens to identify peptides from combinatorial libraries that representspecific anti-tumor, anti-viral, or anti-bacterial epitopes. Relevant tothe chaperone activity of GRP94 and HSP90, activators thereof can alsoameliorate or prevent cellular damage resulting from ischemicconditions.

Inhibitors of GRP94/HSP90 function can possess anti-tumor activity.Inhibitors of GRP94/HSP90 function can also interfere with theprocessing of viral or bacterial proteins in infectious states and slowthe progress of these infections. Inhibitors of GRP94/HSP90 function canalso be administered to a vertebrate subject to decrease theantigenicity of tissues to alleviate transplanted tissue rejection oreven slow the progression of autoimmune diseases such as rheumatoidarthritis and systemic lupus erythramatosis. Inhibitors of GRP94activity can also be used for treatment of diseases, such as cancer, byinhibiting or blocking the egress of proteins (e.g., growth factors)from the endoplasmic reticulum.

A biological activity of a Hsp90 protein such as GRP94 that is modulatedin accordance with the present invention can include, but is not limitedto, loading activity in the formation of a complex with antigenicmolecules, eliciting an immune response in a subject; treating orpreventing a type of cancer in a subject; treating or preventing aninfectious disease in a subject; sensitizing antigen presenting cells(APC), particularly with respect to a type of cancer or an infectiousdisease; and enhancing protein transport along the endoplasmicreticulum.

Another modulatable biological activity of a Hsp90 protein comprisespreventing or ameliorating cellular damage arising from conditions ofischemia/reperfusion including but not limited to cardiac arrest,asystole and sustained ventricular arrythmias, cardiac surgery,cardiopulmonary bypass surgery, organ transplantation, spinal cordinjury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke,cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, anepileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder,Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), or neonatal stress. In this case, a ligand can modulate anendogenous Hsp90 protein by promoting conformational activation of theHsp90 protein. Preferably, the ligand was identified according to ascreening or rational drug design method disclosed herein and isrelevant for the modulation of GRP94 or HSP90.

XIII.A. In vitro Production of GRP94-Antigenic Molecule Complexes

In accordance with the present invention, complexes of an Hsp90 protein,such as GRP94, to antigenic molecules are produced in vitro using anHsp90 protein activity modulator. As will be appreciated by thoseskilled in the art, the peptides either isolated by procedures disclosedherein, chemically synthesized or recombinantly produced, can bereconstituted with a variety of naturally purified or recombinant Hsp90proteins in vitro to generate, for example, immunogenic non-covalentGRP94-antigenic molecule complexes. Alternatively, exogenous antigens orantigenic/immunogenic fragments or derivatives thereof can benon-covalently complexed to an Hsp90 protein for use in theimmunotherapeutic or prophylactic vaccines of the invention. Thecomplexes can then be purified using any suitable method, and arepreferably purified via the affinity purification methods of the presentinvention disclosed herein above.

In a representative approach, antigenic molecules (1 μg) and GRP94 (9μg) are admixed to give an approximately 5 antigenic molecule: 1 GRP94molar ratio. Then, the mixture is incubated for 15 minutes to 3 hours at4° C. to 45° C. with bis-ANS in a quantity equimolar to GRP94 in asuitable binding buffer such as one containing 20 mM sodium phosphate,pH 7.2, 350 mM NaCl, 3 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride(PMSF). The preparations are centrifuged through CENTRICON®10 assembly(Amicon of Beverly, Mass.) to remove any unbound peptide. Theassociation of the peptides with GRP94 can be assayed by SDS-PAGE.Additional representative approaches are disclosed in the Examples.

Following complexing, the immunogenic GRP94-antigenic molecule complexescan optionally be assayed in vitro using, for example, the mixedlymphocyte tumor cell assay (MLTC) described herein. Once immunogeniccomplexes have been isolated they can be optionally characterizedfurther in animal models using the preferred administration protocolsand excipients discussed herein.

XIII.A.1. Exogenous Antigenic Molecules

Antigens or antigenic portions thereof can be selected for use asantigenic molecules, for complexing to an Hsp90 protein, such as GRP94,from among those known in the art or determined by immunoassay to beable to bind to antibody or MHC molecules (antigenicity) or generateimmune response (immunogenicity). To determine immunogenicity orantigenicity by detecting binding to antibody, various immunoassaysknown in the art can be used, including but not limited to competitiveand non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion, assays, in vivo immunoassays (usingcolloidal gold, enzyme or radioisotope labels, for example), westernblots, immunoprecipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmuno-electrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many methods and techniques are known in the art for detectingbinding in an immunoassay and can be used. In one embodiment fordetecting immunogenicity, T cell-mediated responses can be assayed bystandard methods, e.g., in vitro cytotoxicity assays or in vivodelayed-type hypersensitivity assays.

Potentially useful antigens or derivatives thereof for use as antigenicmolecules can also be identified by various criteria, such as theantigen's involvement in neutralization of a pathogen's infectivity(wherein it is desired to treat or prevent infection by such a pathogen)(Norrby (1985) “Summary” in Vaccines 85, Lerner et al. (eds.), pp.388-389, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), type orgroup specificity, recognition by subjects' antisera or immune cells,and/or the demonstration of protective effects of antisera or immunecells specific for the antigen. In addition, where it is desired totreat or prevent a disease caused by a pathogen, the antigen's encodedepitope should preferably display a small or no degree of antigenicvariation in time or amongst different isolates of the same pathogen.

Preferably, where it is desired to treat or prevent cancer, knowntumor-specific antigens or fragments or derivatives thereof are used.For example, such tumor specific or tumor-associated antigens includebut are not limited to KS ¼ pan-carcinoma antigen (Perez & Walker (1990)J Immunol 142:3662-3667; Bumal (1988) Hybridoma 7(4):407-415); ovariancarcinoma antigen (CA125) (Yu et al. (1991) Cancer Res 51(2):468-475);prostatic acid phosphate (Tailer et al. (1990) Nuc Acids Res18(16):4928); prostate specific antigen (Henttu & Vihko (1989) BiochemBiophys Res Comm 160(2):903-910; Israeli et al. (1993) Cancer Res53:227-230); melanoma-associated antigen p97 (Estin et al. (1989) J NatlCancer Inst 81(6):445-446); melanoma antigen gp75 (Vijayasardahl et al.(1990) J Exp Med 171(4):1375-1380); high molecular weight melanomaantigen (Natali et al. (1987) Cancer 59:55-63) and prostate specificmembrane antigen. In a specific embodiment, an antigen or fragment orderivative thereof specific to a certain tumor is selected forcomplexing to an Hsp90 protein, such as GRP94, and subsequentadministration to a subject having that tumor. The term “specific” canrefer to an antigen found in or on a tumor cell, but not in or on anon-tumorous or non-cancerous cell.

Preferably, where it is desired to treat or prevent viral diseases,molecules comprising epitopes of known viruses are used. For example,such antigenic epitopes can be prepared from viruses including, but notlimited to, hepatitis type A hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus (RSV), papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II). Preferably, where it is desired to treat or preventbacterial infections, molecules comprising epitopes of known bacteriaare used. For example, such antigenic epitopes can be prepared frombacteria including, but not limited to, Mycobacteria, Mycoplasma,Neisseria, and Legionella.

Preferably, where it is desired to treat or prevent protozoalinfectious, molecules comprising epitopes of known protozoa are used.For example, such antigenic epitopes can be prepared from protozoaincluding, but not limited to, Leishmania, Kokzidioa, and Trypanosoma.Preferably, where it is desired to treat or prevent parasiticinfectious, molecules comprising epitopes of known parasites are used.For example, such antigenic epitopes can be from parasites including,but not limited to, Chlamydia and Rickettsia.

XIII.A.2. Peptides from MHC Complexes

Candidate immunogenic or antigenic peptides can be isolated from eitherendogenous Hsp90-peptide complexes as described above or from endogenousMHC-peptide complexes for use subsequently as antigenic molecules, bycomplexing in vitro to an Hsp90 protein, such as GRP94. The isolation ofpotentially immunogenic peptides from MHC molecules is well known in theart and so is not described in detail herein. See Falk et al. (1990)Nature 348:248-251; Rotzsche et al. (1990) Nature 348:252-254; Elliottet al. (1990) Nature 348:191-197; Falk et al. (1991) Nature 351:290-296;Demotz et al. (1989) Nature 343:682-684; Rotzsche et al. (1990) Science249:283-287, the disclosures of which are incorporated herein byreference. Briefly, MHC-peptide complexes can be isolated by aconventional immuno-affinity procedure. The peptides can then be elutedfrom the MHC-peptide complex by incubating the complexes in the presenceof about 0.1% TFA in acetonitrile. The eluted peptides can befractionated and purified by HPLC as described herein.

XIII.B. Therapeutic Methods for Modulating Hsp90 Biological Activity

A therapeutic method according to the present invention comprisesadministering to a subject in need thereof a substance that modulates,i.e., inhibits or promotes, biological activity of an Hsp90 protein,such as GRP94. Representative substances, also referred to as “ligandcompositions” or “modulators”, are disclosed herein (e.g., compounds offormula (I)) and can also be identified according to any of thescreening assays set forth herein. The method comprises treating asubject suffering from a disorder wherein modulation of the biologicalactivity of an Hsp90 protein is desirable by administering to thesubject an effective amount of an Hsp90 modulator. Preferably, the Hsp90protein is GRP94. More preferably, the modulator elicits aconformational change in an Hsp90 protein. Even more preferably, themodulator is identified according to a screening assay described herein.

By the term “modulating”, it is meant that the substance can eitherpromote or inhibit the biological activity of an Hsp90 protein,depending on the disorder to be treated, and can affect one or severalof the Hsp90 proteins, including GRP94. Administration can providetreatment of disorders that can be exacerbated by GRP94/HSP90-mediatedmechanisms, including but not limited to, cancer, infectious diseases,and ischemic conditions.

The subject treated in the present invention in its many embodiments isdesirably a human subject, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to invertebrate and to all vertebrate species, includingmammals, which are intended to be included in the term “subject”. Thisis particularly the case in view of the phylogenetically ubiquitousnature of Hsp90 proteins. Moreover, a mammal is understood to includeany mammalian species in which treatment or prevention of cancer orinfectious diseases is desirable, particularly agricultural and domesticmammalian species.

The methods of the present invention are particularly useful in thetreatment of warm-blooded vertebrates. Therefore, the invention concernsmammals and birds.

More particularly, contemplated is the treatment of mammals such ashumans, as well as those mammals of importance due to being endangered(such as Siberian tigers), of economical importance (animals raised onfarms for consumption by humans) and/or social importance (animals keptas pets or in zoos) to humans, for instance, carnivores other thanhumans (such as cats and dogs), swine (pigs, hogs, and wild boars),ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison,and camels), and horses. Also contemplated is the treatment of birds,including the treatment of those kinds of birds that are endangered,kept in zoos, as well as fowl, and more particularly domesticated fowl,i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, andthe like, as they are also of economical importance to humans. Thus,contemplated is the treatment of livestock, including, but not limitedto, domesticated swine (pigs and hogs), ruminants, horses, poultry, andthe like.

In one embodiment, a ligand composition or modulator is administered inconjunction with a complex comprising an Hsp90 protein (preferably GRP94or HSP90) and an antigenic molecule. Preferably, the complex is“autologous” to the subject; that is, the complex is isolated fromeither from the infected cells or the cancer cells or precancerous cellsof the subject (e.g., preferably prepared from infected tissues or tumorbiopsies of a subject). More preferably, the complex is purified inaccordance with a purification method of the present invention disclosedherein above.

Alternatively, the complex is produced in vitro (e.g., wherein a complexwith an exogenous antigenic molecule is desired). Alternatively, theHsp90 protein (preferably GRP94 or HSP90) and/or the antigenic moleculecan be isolated from a particular subject or from others or byrecombinant production methods using a cloned Hsp90 protein (preferablyGRP94 or HSP90) originally derived from a particular subject or fromothers. Exogenous antigens and fragments and derivatives (both peptideand non-peptide) thereof for use in complexing with an Hsp90 protein,can be selected from among those known in the art, as well as thosereadily identified by standard immunoassays know in the art by theability to bind antibody or MHC molecules (antigenicity) or generateimmune response (immunogenicity). Complexes of an Hsp90 protein(preferably GRP94 or HSP90) and antigenic molecules can be isolated fromcancer or precancerous tissue of a subject, or from a cancer cell line,or can be produced in vitro (as is necessary in the embodiment in whichan exogenous antigen is used as the antigenic molecule). Preferably, thecomplex is purified in accordance with a purification method of thepresent invention disclosed herein above.

The invention also provides a method for measuring tumor rejection invivo in a subject, preferably a human subject, comprising measuring thegeneration by the subject of MHC Class I-restricted CD8⁺ cytotoxic Tlymphocytes specific to the tumor after administering a complexcomprising GRP94 and antigenic molecules specific to the tumor inconjunction with an GRP94 biological activity modulator. Preferably,GRP94 comprises human GRP94. The immunogenic GRP94-peptide complexes ofthe invention can include any complex containing a GRP94 and a peptidethat is capable of inducing an immune response in a subject. Thepeptides are preferably non-covalently associated with the GRP94.

Although the Hsp90 protein can be allogenic to the subject (e.g.,isolated from cancerous tissue from a second vertebrate subject that isthe same type as a cancerous tissue present in a first vertebratesubject to be treated), in a preferred embodiment, the Hsp90 protein isautologous to (derived from) the subject to whom they are administered.The Hsp90 protein and/or antigenic molecules can be purified fromnatural sources, chemically synthesized, or recombinantly produced.Preferably, the complex and/or antigenic molecule is purified inaccordance with a purification method of the present invention disclosedherein above. The invention provides methods for determining doses forhuman cancer immunotherapy by evaluating the optimal dose of an Hsp90protein non-covalently bound to peptide complexes in experimental tumormodels and extrapolating the data. Specifically, a scaling factor notexceeding a fifty-fold increase over the effective dose estimated inanimals, is used as the optimal prescription method for cancerimmunotherapy or vaccination in human subjects. Preferably, the Hsp90protein is GRP94.

The invention provides combinations of compositions that enhance theimmunocompetence of the host individual and elicit specific immunityagainst infectious agents or specific immunity against preneoplastic andneoplastic cells. The therapeutic regimens and pharmaceuticalcompositions of the invention are described below. These compositionshave the capacity to prevent the onset and progression of infectiousdiseases and prevent the development of tumor cells and to inhibit thegrowth and progression of tumor cells, indicating that such compositionscan induce specific immunity in infectious diseases and cancerimmunotherapy. For example, Hsp90-antigenic molecule complexes can beadministered in combination with other complexes, such as calreticulin,and antigenic molecules in accordance with the methods of the presentinvention.

Accordingly, the invention provides methods of preventing and treatingcancer in a subject. A representative method comprises administering atherapeutically effective amount of an Hsp90 modulator (preferably aGRP94 modulator) to a subject in need thereof. Such a subject caninclude but is not limited to a subject suffering from cancer or at riskto develop cancer. Representative modulators that can be employed in themethod comprise ligands that inhibit GRP94 (Hsp90) function. Suchligands are designed and identifed using the screening methods disclosedherein and are thus employed as anti-tumor drugs, and/or anti-neoplasticagents. Characterization of these activities can be accomplished viatechniques disclosed herein and known in the art.

In another embodiment, the method comprises administering a complexcomprising an Hsp90 protein and pertinent antigenic molecule inconjunction with a modulator that stimulates the immunocompetence of thehost individual and elicits specific immunity against the preneoplasticand/or neoplastic cells. Preferably, the Hsp90 protein is GRP94.

As used herein, “preneoplastic” cell refers to a cell which is intransition from a normal to a neoplastic form; and morphologicalevidence, increasingly supported by molecular biologic studies,indicates that preneoplasia progresses through multiple steps.Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia,or most particularly, dysplasia (for review of such abnormal growthconditions. See Robbins & Angell (1976) Basic Pathology, 2d Ed., pp.68-79, W. B. Saunders Co., Philadelphia, Pa.).

Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder. Althoughpreneoplastic lesions can progress to neoplasia, they can also remainstable for long periods and can even regress, particularly if theinciting agent is removed or if the lesion succumbs to an immunologicalattack by its host.

The therapeutic regimens and pharmaceutical compositions of theinvention can be used with additional adjuvants or biological responsemodifiers including, but not limited to, the cytokines IFN-α, IFN-γ,IL-2, IL-4, IL-6, TNF, or other cytokine affecting immune cells. Inaccordance with this aspect of the invention, a complex of an Hsp90protein and an antigenic molecule along with a modulator areadministered in combination therapy with one or more of these cytokines.Preferably, the Hsp90 protein is GRP94.

The invention also pertains to administration of a complex of an Hsp90protein and an antigenic molecule and a modulator to individuals atenhanced risk of cancer due to familial history or environmental riskfactors. Preferably, the Hsp90 protein is GRP94.

Enzymes in the cells of higher eukaryotes that mediate the steady stateand stress-elicited production of a native GRP94 ligand can also bemodulated in accordance with the present invention. Particularly, suchcatabolic enzymes represent appropriate and rational targets formodulation to elicit an increase in the steady state levels of a nativeGRP94 ligand and thereby lead to the elicitation of the structural andfunctional activation of chaperone and peptide binding activity of GRP94disclosed herein.

Protein misfolding disorders are a common component of numerous geneticdisease states including, but not limited to, cystic fibrosis, familialhypercholesterolemia, retinitis pigmentosa and α1-antitrypsinmisfolding. Compounds that modulate the activity of the Hsp90 family ofmolecular chaperones can thus be used in accordance with a therapeuticmethod of the present invention for reversing the protein foldingdefects that identify the disease state or for enhancing proteintransport from the endoplasmic reticulum of a cell. Thus, a compoundthat modulates the conformation of GRP94 can be used to treat a diseasestate resulting from defects in protein transport into or from theendoplasmic reticulum. Compounds that abrogate GRP94 activity can beused for the treatment of a disease state, such as cancer, wherein atherapeutic benefit can be provided by blocking the egress of proteins(e.g., growth factors) from the endoplasmic reticulum. conversely,compounds that promote GRP94 activity can be used to treat a diseasewherein a therapeutic benefit can be provided by enhancing proteinexport from the endoplasmic reticulum.

The present invention also pertains to administration of compounds forthe prevention or amelioration of cellular damage arising fromconditions of ischemia/reperfusion including but not limited to cardiacarrest, asystole and sustained ventricular arrythmias, cardiac surgery,cardiopulmonary bypass surgery, organ transplantation, spinal cordinjury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke,cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, anepileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder,Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), or neonatal stress. In one embodiment, a composition comprising aHsp90 ligand is administered to promote conformational activation of aHsp90 protein, thereby promoting its cellular protective functionrelevant to recovery following a injury or onset of a disease stateassociated with ischemia. In another embodiment, administration of acomposition comprising a Hsp90 ligand can alter a subsequent cellularresponse to an ischemic condition at a tissue location in a subject.Cells at the tissue location are contacted with a Hsp90 protein ligand,whereby Hsp90 activity in the cells is enhanced to a degree effective toalter a subsequent cellular response to an ischemic condition.Preferably, the therapeutic composition comprises a ligand identifiedaccording to a screening or rational drug design method disclosedherein. Also preferably, the therapeutic composition modulates theactivity of GRP94 or HSP90.

XIII.C. Dosage Regimens

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to administer anamount of the active compound(s) that is effective to achieve thedesired therapeutic response for a particular subject. The selecteddosage level will depend upon the activity of the particular compound,the route of administration, the severity of the condition beingtreated, and the condition and prior medical history of the subjectbeing treated. However, it is within the skill of the art to start dosesof the compound at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. If desired, the effective daily dose may bedivided into multiple doses for purposes of administration, e.g., two tofour separate doses per day. It will be understood, however, that thespecific dose level for any particular subject will depend upon avariety of factors including the body weight, general health, diet, timeand route of administration, combination with other drugs and theseverity of the particular disease being treated.

The dosage ranges for the administration of a modulator depend upon theform of the modulator, and its potency, as described further herein, andare amounts large enough to produce the desired effect. The dosageshould not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can also be adjusted by the individualphysician in the event of any complication.

The therapeutic compositions can be administered as a unit dose. Theterm “unit dose” when used in reference to a therapeutic compositionemployed in the method of the present invention refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent; i.e., carrier or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies can also be administered.

A therapeutically effective amount is an amount of a modulatorsufficient to produce a measurable modulation of Hsp90 protein(preferably GRP94) biological activity in a subject being treated, i.e.,an Hsp90 protein biological activity-modulating amount. Modulation ofHsp90 protein biological activity can be measured using the screeningmethods disclosed herein, via the method disclosed in the Examples, orby other methods known to one skilled in the art.

The potency of a modulator can vary, and therefore a “therapeuticallyeffective” amount can vary. However, as shown by the present assaymethods, one skilled in the art can readily assess the potency andefficacy of a candidate modulator of this invention and adjust thetherapeutic regimen accordingly. A modulator of Hsp90 protein(preferably GRP94) biological activity can be evaluated by a variety ofmethods and techniques including the screening assays disclosed herein.

A preferred modulator has the ability to substantially bind an Hsp90protein in solution at modulator concentrations of less than one (1)micromolar (μM), preferably less than 0.1 μM, and more preferably lessthan 0.01 μM. By “substantially” is meant that at least a 50 percentreduction in biological activity is observed by modulation in thepresence of the modulator, and at 50% reduction is referred to herein asan “IC50 value”.

In one embodiment, the therapeutically effective amount of a modulatorcan respectively range from about 0.01 mg to about 10,000 mg per day.Alternatively, the therapeutically effective amount of a modulator canrespectively range from about 0.1 mg to about 1,000 mg per day.Alternatively, the therapeutically effective amount of a modulator canrespectively range from about 1 mg to about 300 mg per day. In apreferred embodiment, the therapeutically effective amount of amodulator can respectively range from about 15 mg per kg body weight perday to about 35 mg per kg body weight per day.

It was established in experimental tumor models (Blachere et al., 1993)that the lowest dose of heat shock proteins noncovalently bound topeptide complexes which produced tumor regression in mice was between 10and 25 microgram/mouse weighing 20-25 g which is equal to 25 mg/25 g=1mg/kg. Conventional methods extrapolate to human dosages based on bodyweight and surface area. For example, conventional methods ofextrapolating human dosage based on body weight can be carried out asfollows: since the conversion factor for converting the mouse dosage tohuman dosage is Dose Human per kg=Dose Mouse per kg×12 (Freireich et al.(1966) Cancer Chemotherap Rep 50:219-244), the effective dose ofHsp90-peptide complexes in humans weighing 70 kg should be 1mg/kg÷12×70, i.e., about 6 mg (5.8 mg).

Drug doses are also given in milligrams per square meter of body surfacearea because this method rather than body weight achieves a goodcorrelation to certain metabolic and excretionary functions (Shirkey(1965) JAMA 193:443). Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich et al. (1966) CancerChemotherap Rep 50:219-244. Briefly, to express a mg/kg dose in anygiven species as the equivalent mg/sq m dose, multiply the dose by theappropriate km factor. In adult human, 100 mg/kg is equivalent to 100mg/kg×37 kg/sq m=3700 mg/sq m.

International Publication Nos. WO 95/24923, WO 97/10000, WO 97/10002,and WO 98/34641, as well as U.S. Pat. Nos. 5,750,119, 5,830,464, and5,837,251, each provide dosages of the purified complexes of heat shockproteins and antigenic molecules, and the entire contents of each ofthese documents are herein incorporated by reference. Briefly, and asapplied to the present invention, an amount of Hsp90 protein (preferablyGRP94)-antigenic molecule complexes is administered that is in the rangeof about 10 microgram to about 600 micrograms for a human subject, thepreferred human dosage being the same as used in a 25 g mouse, i.e., inthe range of 10-100 micrograms. The dosage for Hsp90 protein (preferablyGRP94)-peptide complexes in a human subject provided by the presentinvention is in the range of about 50 to 5,000 micrograms, the preferreddosage being 100 micrograms.

In a series of preferred and more preferred embodiments, theHsp90-peptide complex is administered in an amount of less than about 50micrograms. In this case, the Hsp90 protein (preferably GRP94)-peptidecomplex is preferably administered in an amount of ranging from about 5to about 49 micrograms. In a preferred embodiment, a GRP94-peptidecomplex is administered in an amount of less than about 10 micrograms.In this case, the GRP94-peptide complex is preferably administered in anamount ranging from about 0.1 to about 9.0 micrograms. More preferably,the GRP94-peptide complexes is administered in an amount ranging fromabout 0.5 to about 2.0 micrograms. In accordance with one aspect of thepresent invention, administration of a lower dosage of complex isfacilitated and preferred when a modulator is also administered.

The doses recited above are preferably given once weekly for a period ofabout 4-6 weeks, and the mode or site of administration is preferablyvaried with each administration. In a preferred example, subcutaneousadministrations are given, with each site of administration variedsequentially. For example, half the dose can be given in one site andthe other half on an other site on the same day.

Alternatively, the mode of administration is sequentially varied. Forexample, weekly injections are given in sequence subcutaneously,intramuscularly, intravenously or intraperitoneally. After 4-6 weeks,further injections are preferably given at two-week intervals over aperiod of time of one month. Later injections can be given monthly. Thepace of later injections can be modified, depending upon the subject'sclinical progress and responsiveness to the immunotherapy.

XIII.D. Therapeutic Compositions for Immune Responses to Cancer

Compositions comprising an Hsp90 protein bound (e.g., GRP94-preferablynon-covalently bound) to antigenic molecules are administered to elicitan effective specific immune response to the complexed antigenicmolecules (and preferably not to the HSP90 protein). In a preferredembodiment, non-covalent complexes of the Hsp90 protein with peptidesare prepared and purified postoperatively from tumor cells obtained fromthe cancer patient that have also been treated with an Hsp90 proteinbiological activity modulator in accordance with the present invention.A preferred Hsp90 protein is GRP94. In a more preferred embodiment, thecomplexes are purified using an affinity purification method of thepresent invention, as disclosed herein above.

In accordance with the methods described herein, immunogenic orantigenic peptides that are endogenously complexed to Hsp90 (e.g. GRP94)or MHC antigens can be used as antigenic molecules. For example, suchpeptides can be prepared that stimulate cytotoxic T cell responsesagainst different tumor antigens (e.g., tyrosinase, gp100, melan-A,gp75, mucins, etc.) and viral proteins including, but not limited to,proteins of immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), hepatitis type A, hepatitistype B, hepatitis type C, influenza, varicella, adenovirus, herpessimplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,rhinovirus, echovirus, rotavirus, respiratory syncytial virus (RSV),papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virusand polio virus. In the embodiment wherein the antigenic molecules arepeptides noncovalently complexed to GRP94 in vivo, the complexes can beisolated from cells, or alternatively, produced in vitro from purifiedpreparations each of GRP94 and antigenic molecules. The complexes can befurther purified using an affinity purification method of the presentinvention, as disclosed herein above.

In another specific embodiment, antigens of cancers (e.g., tumors) orinfectious agents (e.g., viral antigen, bacterial antigens, etc.) can beobtained by purification from natural sources, by chemical synthesis, orrecombinantly, and, through in vitro procedures such as those describedherein, complexed to GRP94. The complexes can also be further purifiedusing an affinity purification method of the present invention, asdisclosed herein above.

XIII.E. Formulation

In accordance with the present invention, modulators as well asantigenic molecule complexes can be formulated into pharmaceuticalpreparations for administration to a subject for treatment or preventionof cancer or infectious diseases. Compositions comprising a complexprepared in accordance with the present invention are formulated in acompatible pharmaceutical carrier can be prepared, packaged, and labeledfor treatment of the indicated disorder (e.g. cancer or infectiousdisease).

If the modulator or complex is water-soluble, then it can be formulatedin an appropriate buffer, for example, phosphate buffered saline orother physiologically compatible solutions. Alternatively, if amodulator or a resulting complex has poor solubility in aqueoussolvents, then it can be formulated with a non-ionic surfactant, such asTWEEN™, or polyethylene glycol. Thus, the compounds and theirphysiologically acceptable solvates can be formulated for administrationby inhalation or insufflation (either through the mouth or the nose) ororal, buccal, parenteral, rectal administration or, in the case oftumors, directly injected into a solid tumor.

For oral administration, the pharmaceutical preparation can be in liquidform, for example, solutions, syrups or suspensions, or can be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions can take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets can be coated by methodswell-known in the art. Preparations for oral administration can besuitably formulated to give controlled release of the active compound.

For buccal administration, the compositions can take the form of tabletsor lozenges formulated in conventional manner. For administration byinhalation, the compounds for use according to the present invention areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compositions can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, for example, inampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well known examplesof delivery vehicles or carriers for hydrophilic drugs.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically or prophylactically effective amounts of a modulatorand/or a antigenic molecule complex in pharmaceutically acceptable form.The modulator and the antigenic molecule complex in a vial of a kit ofthe invention can be in the form of a pharmaceutically acceptablesolution, e.g., in combination with sterile saline, dextrose solution,or buffered solution, or other pharmaceutically acceptable sterilefluid. Alternatively, the modulator or complex can be lyophilized ordesiccated; in this instance, the kit optionally further comprises in acontainer a pharmaceutically acceptable solution (e.g., saline, dextrosesolution, etc.), preferably sterile, to reconstitute the modulatorcomplex to form a solution for injection purposes.

In another embodiment, a kit of the invention further comprises needlesor syringes, preferably packaged in sterile form, for injecting themodulator and complex, and/or a packaged alcohol pad. Instructions areoptionally included for administration of antigenic molecule complexesby a clinician or by the subject.

XIV. Target Infectious Diseases

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents including, but notlimited to, viruses, bacteria, fungi, protozoa and parasites. In oneembodiment of the present invention wherein it is desired to treat asubject having an infectious disease, the above-described affinitypurification methods are used to isolate GRP94-peptide complexes fromcells infected with an infectious organism, e.g., of a cell line or froma subject. Thus, preferably, the peptides are found in cells infectedwith an infectious organism and not in cells that are not infected.

Viral diseases that can be treated or prevented by the methods of thepresent invention include, but are not limited to, those caused byhepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus (RSV), papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

Bacterial diseases that can be treated or prevented by the methods ofthe present invention are caused by bacteria including, but not limitedto, Mycobacteria, Mycoplasma, Neisseria, and Legionella.

Protozoal diseases that can be treated or prevented by the methods ofthe present invention are caused by protozoa including, but not limitedto, Leishmania, Kokzidioa, and Trypanosoma. Parasitic diseases that canbe treated or prevented by the methods of the present invention arecaused by parasites including, but not limited to, Chlamydia andRickettsia.

XV. Target Cancers

Cancers that can be treated or prevented by the methods of the presentinvention include, but not limited to human sarcomas and carcinomas,including but not limited to fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenströom's macroglobulinemia, and heavychain disease.

In a specific embodiment the cancer is metastatic. In another specificembodiment, the subject having a cancer is immunosuppressed by reason ofhaving undergone anti-cancer therapy (e.g., chemotherapy radiation)prior to administration of the GRP94-antigenic molecule complexes and aGRP94 modulator in accordance with the present invention.

XVI. Combination With Adoptive Immunotherapy

Adoptive immunotherapy refers to a therapeutic approach for treatingcancer or infectious diseases in which immune cells are administered toa host with the aim that the cells mediate either directly or indirectlyspecific immunity to tumor cells and/or antigenic components orregression of the tumor or treatment of infectious diseases, as the casecan be. In accordance with the methods described herein, APC aresensitized with GRP94 preferably noncovalently complexed with antigenic(or immunogenic) molecules in conjunction with a GRP94 biologicalactivity modulator and used in adoptive immunotherapy.

According to one embodiment of the present invention, therapy byadministration of GRP94-peptide complexes and a GRP94 biologicalactivity modulator, using any desired route of administration, iscombined with adoptive immunotherapy using APC sensitized withGRP94-antigenic molecule complexes and a modulator. The sensitized APCcan be administered concurrently with GRP94-peptide complexes and themodulator, or before or after administration of GRP94-peptide complexesand the modulator. Furthermore, the mode of administration can bevaried, including but not limited to, e.g., subcutaneously,intravenously, intraperitoneally, intramuscularly, intradermally ormucosally.

XVI.A. Obtaining Macrophages and Antigen-Presenting Cells

The antigen-presenting cells, including but not limited to macrophages,dendritic cells and B-cells, are preferably obtained by production invitro from stem and progenitor cells from human peripheral blood or bonemarrow as described by Inaba (1992) J Exp Med 176:1693-1702.

APC can be obtained by any of various methods known in the art. In apreferred aspect human macrophages are used, obtained from human bloodcells. By way of example but not limitation, macrophages can be obtainedas follows: mononuclear cells are isolated from peripheral blood of asubject (preferably the subject to be treated), by Ficoll-Hypaquegradient centrifugation and are seeded on tissue culture dishes whichare pre-coated with the subject's own serum or with other AB+ humanserum. The cells are incubated at 37° C. for 1 hr, then non-adherentcells are removed by pipetting. To the adherent cells left in the dish,is added cold (4° C.) 1 mM EDTA in phosphate-buffered saline and thedishes are left at room temperature for 15 minutes. The cells areharvested, washed with RPMI buffer and suspended in RPMI buffer.Increased numbers of macrophages can be obtained by incubating at 37° C.with macrophage-colony stimulating factor (M-CSF); increased numbers ofdendritic cells can be obtained by incubating withgranulocyte-macrophage-colony stimulating factor (GM-CSF) as describedin detail by Inaba, et al. (1992).

XVI.B. Sensitization of Macrophages and Antigen Presenting Cells WithGRP94-Peptide Complexes

APC are sensitized with GRP94 (preferably noncovalently) bound toantigenic molecules by incubating the cells in vitro with the complexesand a modulator. The APC are sensitized with complexes of GRP94 andantigenic molecules preferably by incubating in vitro with theGRP94-complex and a modulator at 37° C. for 15 minutes to 24 hours. Byway of example but not limitation, 4×10⁷ macrophages can be incubatedwith 10 microgram GRP94-peptide complexes per ml or 100 microgramGRP94-peptide complexes per mL and a modulator in an equimolar amountwith respect to the GRP94-peptide complex at 37° C. for 15 minutes-24hours in 1 mL plain RPMI medium. The cells are washed three times andresuspended in a physiological medium preferably sterile, at aconvenient concentration (e.g., 1×10⁷/ml) for injection in a subject.Preferably, the subject into which the sensitized APCs are injected isthe subject from which the APC were originally isolated (autologousembodiment).

Optionally, the ability of sensitized APC to stimulate, for example, theantigen-specific, class I-restricted cytotoxic T-lymphocytes (CTL) canbe monitored by their ability to stimulate CTLs to release tumornecrosis factor, and by their ability to act as targets of such CTLs.

XVI.C. Reinfusion of Sensitized APC

The sensitized APC are reinfused into the subject systemically,preferably intravenously, by conventional clinical procedures. Theseactivated cells are reinfused, preferentially by systemic administrationinto the autologous subject. Subjects generally receive from about 10⁶to about 10¹² sensitized macrophages, depending on the condition of thesubject. In some regimens, subjects can optionally receive in addition asuitable dosage of a biological response modifier including but notlimited to the cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF or othercytokine growth factor.

XVII. Autologous Embodiment

The specific immunogenicity of an Hsp90 protein derives not from Hsp90protein per se, but from the peptides bound to them. In a preferredembodiment of the invention directed to the use of autologous complexesof GRP94-peptides as cancer vaccines wherein the immunogenicity has beenenhanced with a modulator in accordance with the present invention, twoof the most intractable hurdles to cancer immunotherapy arecircumvented. First is the possibility that human cancers, like somecancers of experimental animals, are antigenically distinct. Thus, in anembodiment of the present invention, GRP94 chaperones antigenic peptidesof the cancer cells from which they are derived and circumvent thishurdle.

Second, most current approaches to cancer immunotherapy focus ondetermining the CTL-recognized epitopes of cancer cell lines. Thisapproach requires the availability of cell lines and CTLs againstcancers. These reagents are unavailable for an overwhelming proportionof human cancers. Thus, in an embodiment of the present inventiondirected to autologous complexes of GRP94 and peptides, preferablywherein the immunogenicity has been enhanced with a modulator of thepresent invention, cancer immunotherapy does not depend on theavailability of cell lines or CTLs nor does it require definition of theantigenic epitopes of cancer cells. These advantages make autologousHsp90 proteins (e.g., GRP94) noncovalently bound to peptide complexesattractive and novel immunogens against cancer.

XVIII. Prevention and Treatment of Primary and Metastatic NeoplasticDiseases

There are many reasons why immunotherapy as provided by the presentinvention is desired for use in cancer patients. First, if cancerpatients are immunosuppressed and surgery, with anesthesia, andsubsequent chemotherapy, can worsen the immunosuppression, then withappropriate immunotherapy in the preoperative period, thisimmunosuppression can be prevented or reversed. This could lead to fewerinfectious complications and to accelerated wound healing. Second, tumorbulk is minimal following surgery and immunotherapy is most likely to beeffective in this situation. A third reason is the possibility thattumor cells are shed into the circulation at surgery and effectiveimmunotherapy applied at this time can eliminate these cells.

The preventive and therapeutic methods of the invention are directed atenhancing the immunocompetence of the cancer patient either beforesurgery, at or after surgery, and to induce tumor-specific immunity tocancer cells (i.e., an immune response against the cancer cells but nota non-cancerous or normal cell), with the objective being inhibition ofcancer, and with the ultimate clinical objective being total cancerregression and eradication.

XIX. Monitoring of Effects During Cancer Prevention and Immunotherapywith Hsp90 Protein-Antigenic Molecule Complexes

The effect of immunotherapy with GRP94-antigenic molecule complexes ondevelopment and progression of neoplastic diseases can be monitored byany methods known to one skilled in the art, including but not limitedto measuring: 1) delayed hypersensitivity as an assessment of cellularimmunity; 2) activity of cytolytic T-lymphocytes in vitro; 3) levels oftumor specific antigens, e.g., carcinoembryonic (CEA) antigens; 4)changes in the morphology of tumors using techniques such as a computedtomographic (CT) scan; 5) changes in levels of putative biomarkers ofrisk for a particular cancer in individuals at high risk, and 6) changesin the morphology of tumors using a sonogram.

Delayed Hypersensitivity Skin Test. Delayed hypersensitivity skin testsare of great value in the overall immunocompetence and cellular immunityto an antigen. Inability to react to a battery of common skin antigensis termed anergy (Sato et al. (1995) Clin Immunol Pathol 74:35-43).Proper technique of skin testing requires that the antigens be storedsterile at 4° C., protected from light and reconstituted shortly beforeuse. A 25- or 27-gauge needle ensures intradermal, rather thansubcutaneous, administration of antigen. Twenty-four and forty-eighthours after intradermal administration of the antigen, the largestdimensions of both erythema and induration are measured with a ruler.Hypoactivity to any given antigen or group of antigens is confirmed bytesting with higher concentrations of antigen or, in ambiguouscircumstances, by a repeat test with an intermediate concentration.

Activity of Cytolytic T-lymphocytes In vitro. 8×10⁶ peripheral bloodderived T lymphocytes isolated by the Ficoll-Hypaque centrifugationgradient technique, are restimulated with 4×10⁴ mitomycin C treatedtumor cells in 3 ml RPMI medium containing 10% fetal calf serum. In someexperiments, 33% secondary mixed lymphocyte culture supernatant or IL-2,is included in the culture medium as a source of T cell growth factors.

In order to measure the primary response of cytolytic T-lymphocytesafter immunization, T cells are cultured without the stimulator tumorcells. In other experiments, T cells are restimulated with antigenicallydistinct cells. After six days, the cultures are tested for cytotoxicityin a 4 hour ⁵¹Cr-release assay. The spontaneous ⁵¹Cr-release of thetargets should reach a level less than 20%. For the anti-MHC class Iblocking activity, a tenfold concentrated supernatant of W6/32 hybridomais added to the test at a final concentration of about 12.5% (Heike etal. (1994) J Immunotherapy 15:165-174).

Levels of Tumor Specific Antigens. Although it can not be possible todetect unique tumor antigens on all tumors, many tumors display antigensthat distinguish them from normal cells. Monoclonal antibody reagentshave permitted the isolation and biochemical characterization of theantigens and have been invaluable diagnostically for distinction oftransformed from nontransformed cells and for definition of the celllineage of transformed cells. The best-characterized humantumor-associated antigens are the oncofetal antigens. These antigens areexpressed during embryogenesis, but are absent or very difficult todetect in normal adult tissue. The prototype antigen is carcinoembryonicantigen (CEA), a glycoprotein found on fetal gut an human colon cancercells, but not on normal adult colon cells. Since CEA is shed from coloncarcinoma cells and found in the serum, it was originally thought thatthe presence of this antigen in the serum could be used to screensubjects for colon cancer. However, subjects with other tumors, such aspancreatic and breast cancer, also have elevated serum levels of CEA.Therefore, monitoring the fall and rise of CEA levels in cancer patientsundergoing therapy has proven useful for predicting tumor progressionand responses to treatment.

Several other oncofetal antigens have been useful for diagnosing andmonitoring human tumors, e.g., alpha-fetoprotein, an alpha-globulinnormally secreted by fetal liver and yolk sac cells, is found in theserum of subjects with liver and germinal cell tumors and can be used asa matter of disease status.

Computed Tomographic (CT) Scan. CT remains the choice of techniques forthe accurate staging of cancers. CT has proved more sensitive andspecific than any other imaging techniques for the detection ofmetastases.

Measurement of Putative Biomarkers. The levels of a putative biomarkerfor risk of a specific cancer are measured to monitor the effect ofGRP94 noncovalently bound to peptide complexes. For example, inindividuals at enhanced risk for prostate cancer, serumprostate-specific antigen (PSA) is measured by the procedure describedby Brawer et al. (1992) J Urol 147:841-845 and Catalona et al. (1993)JAMA 270:948-958; or in individuals at risk for colorectal cancer CEA ismeasured as described above; and in individuals at enhanced risk forbreast cancer, 16—hydroxylation of estradiol is measured by theprocedure described by Schneider et al. (1982) Proc Natl Acad Sci USA79:3047-3051. The references cited above are incorporated by referenceherein in their entirety.

Sonogram. A Sonogram remains an alternative choice of technique for theaccurate staging of cancers.

XX. Target Disorders/Traumas Associated with Ischemia

The present invention provides methods for treating and preventingischemia-induced damage comprising administering a Hsp90 proteinmodulator to a subject wherein Hsp90 activity modulation is desired. Theterm “ischemia”, as used herein, is a loss of blood flow to a tissue.Blood loss is characterized by deprivation of both oxygen and glucose,and leads to ischemic necrosis or infarction. Thus, the term “ischemia”refers to both conditions of oxygen deprivation and of nutrientdeprivation. Loss of blood flow to a particular vascular region isdescribed as “focal ischemia”. Loss of blood flow to an entire tissue orbody is referred to as “global ischemia”.

The present invention provides therapeutic compositions and methods toameliorate cellular damage arising from conditions ofischemia/reperfusion including but not limited to cardiac arrest,asystole and sustained ventricular arrythmias, cardiac surgery,cardiopulmonary bypass surgery, organ transplantation, spinal cordinjury, head trauma, stroke, thromboembolic stroke, hemorrhagic stroke,cerebral vasospasm, hypotension, hypoglycemia, status epilepticus, anepileptic seizure, anxiety, schizophrenia, a neurodegenerative disorder,Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), neonatal stress, and any condition in which a neuroprotectantcomposition that prevents or ameliorates ischemic cerebral damage isindicated, useful, recommended, or prescribed.

The destructive effects of ischemia/reperfusion are manifest as acascade of deleterious events that lead to cell death and ultimatelyorgan failure. The metabolic events underlying ischemia-induced celldeath include energy failure through ATP depletion, cellular acidosis,glutamate release, calcium ion influx, stimulation of membranephospholipid degradation and subsequent free-fatty-acid accumulation,and free radical degeneration. Further, in contrast to apoptotic celldeath, ischemia-induced cell death is characterized by degeneration ofthe most distal cell regions, and subsequent progressive degeneration ofthe cell soma and nucleus (Yamamoto et al. (1986) Brain Res 384:1-10;Yamamoto et al. (1990) Acta Neuropathol 80:487-492). Consistent withthis degeneration profile, cells that bear extended processes, such asneuronal cells, are particularly sensitive to ischemic damage. Althoughnot intended to be limited according to any particular theory, theseobservations suggest that intracellular transport and proteinavailability are essential components of cellular response to stress,and further implicate molecular components of such function, includingHsp90 proteins, as targets for ischemic response.

Thus, in one embodiment, the present invention pertains to the treatmentof central nervous system ischemia. Examples of central nervous systemischemia include cerebral ischemic and spinal column ischemia.

“Cerebral ischemia” is the interruption or reduction of blood flow inthe arteries in or leading to the brain, usually as a result of a bloodclot (thrombus) or other matter (embolus) occluding the artery.

A therapeutic composition of the present invention for the prevention oramelioration of ischemia-induced damage comprises a Hsp90 proteinligand. Preferably, such modulators promote or stabilize an activestructural conformation of an endogenous Hsp90 protein. Also preferably,the Hsp90 ligand modulates the activity of GRP94 or HSP90. Desiredproperties of a composition having a cellular protectant effect includethe following: (1) easy administration by oral or injectable routes(e.g., not significantly degraded in the stomach, intestine, or vascularsystem such that it reaches the tissues to be treated in atherapeutically effective amount), (2) therapeutic activity (e.g.,efficacy) when administered following an ischemic insult, and (3)minimal or no side effects including impairment of cognition, disruptionof motor performance, sedation, hyperexcitability, neuronalvacuolization, and impaired cardiovascular activity.

Compositions comprising Hsp90 protein ligands can be administeredimmediately following a trauma or other event that induces an ischemiccondition. Alternatively, such a composition may be administeredcontinuously or intermittently following detection of a progressivedisorder, including but not limited to neurodegenerative diseases. Instill another embodiment, such a composition may be administered toprevent or improve recovery from a subsequent ischemic condition. Ineach case, effective dose and administration profiles can be determinedusing standard experiments directed at such determination in animalmodels of ischemic conditions as disclosed in, for example, Tacchini etal. (1997) Hepatology 26(1):186-191 and U.S. Pat. Nos. 4,968,671,5,504,090, and 5,733,916. Exemplary animal models are described hereinbelow.

In another embodiment, the present invention pertains to treatment oftissue prior to transplantation. Such tissue is entirely devascularizedfollowing removal from the donor body. A therapeutic compositioncomprising a Hsp90 protein ligand can promote recovery and health of thetransplanted tissue. Several methods for providing such a compound todonor or transplanted tissue are known in the art, including but notlimited to administering the therapeutic compound that promotes organpreservation and health to a donor subject prior to procurance of theorgan, perfusing an isolated organ with the therapeutic composition, andadministering the composition to a transplant recipient prior,concurrent, or following tissue transplantation. See Mizoe et al. (1997)J Surg Res 73(2):160-165 and U.S. Pat. Nos. 5,066,578; 5,756,492; and6,080,730.

In still another embodiment, a composition comprising a Hsp90 proteinmodulator can be repititiously provided to a subject in the absence ofan ischemiccondition, whereby the ability of the subject to tolerate asubsequent ischemic condition is enhanced. Therapeutic compositionscomprising a Hsp90 ligand of the present invention can provide such acellular protectant effect. Preferably, a dose of the therapeuticcomposition intended to induce ischemic tolerance would effect a mildischemic condition as disclosed, for example, in Chen et al. (1996) JCereb Blood Flow Metab 16:566-577 and U.S. Pat. Nos. 5,504,090 and5,733,916.

XX.A. In Vivo Models of Ischemia

Numerous models of ischemic injury and disease are available forevaluating the therapeutic capacity of compositions comprising Hsp90protein modulators. In addition to animal models described herein below,see also Massa et al. (1996) “The Stress Gene Response in Brain” inCerebrovascular and Brain Metabolism Reviews, pp. 95-158,Lippincott-Raven Publishers, Philadelphia, Pa. and references citedtherein. One skilled in the art will appreciate that alternative modelscan be used as disclosed. To assess therapeutic capacity, candidatecompounds can be administered, for example, as a single dose givenintraperitoneally immediately or 30 minutes after reinstating bloodflow.

Transient Global Cerebral Ischemia. U.S. Pat. No. 5,571,840 discloses adog model of cardiac arrest. According to this model, adult dogs areanesthetised and mechanically ventilated to maintain surgical anesthesiaand suppression of corneal reflexes. Expired CO₂ tension and esophagealtemperature are stably maintained before arrest and for at least onehour after resuscitation. Two venous catheters are inserted; one passedby way of the left external jugular vein to the right atrium foradministration of resuscitation drugs, and the other into a muscularbranch of the left femoral vein for fluid administration. Arterial bloodpressure is measured through a catheter placed in a muscular branch ofthe left femoral vein for fluid administration. Arterial blood pressureis measured through a catheter placed in a muscular branch of the leftfemoral artery. Subcutaneous disk electrodes are placed to monitor anelectrocardiogram (ECG).

Each animal is intravenously hydrated before arrest and during recovery.All catheters and electrical leads are passed subcutaneously to exit theskin in the dorsal midscapular region for later attachment to a dogjacket and hydraulic/electric swivel. Pulsatile and mean arterial bloodpressure (MAP), ECG, and end-expiratory CO₂ can be continuously recordedon a six-channel oscillograph. At the conclusion of surgicalinstrumentation, anesthesia is discontinued and ventilation proceedswith room air. When corneal reflexes are apparent, the heart isfibrillated by delivering a 10-15 second, 60 Hz, 2 msec square-wavestimulus to the left ventricular epicardium. Ventilation is discontinuedand circulatory arrest is confirmed by ECG, MAP, and direct observationof the heart. After 9 minutes of normothermic ventricular fibrillation,ventilation is restored and direct cardiac massage is maintained MAPabove 75 mmHg. Mechanical ventilation is continued until spontaneousventilation ensues, but for not longer than 6 hours (typically only 30minutes).

Conditions of stroke can be approximated by occlusion of the primaryarteries to the brain. In one model, a bilateral common carotid arteryocclusion is performed in the gerbil as further disclosed in Karpiak etal. (1989) Ann Rev Pharmacol Toxicol 29:403, Ginsberg & Busto (1989)Stroke 20:1627, and U.S. Pat. No. 6,017,965. Briefly, blood flow to thebrain is interrupted for 7 minutes by clamping the carotid arteries.During the course of these experiments, the core body temperature of theanimals is maintained at 37° C. to prevent a hypothermic reaction.

Permanent Focal Cerebral Ischemia. In another model of cerebralischemia, the middle cerebral artery is occluded in rat as disclosed inKarpiak et al. (1989) Ann Rev Pharmacol Toxicol 29:403, Ginsberg & Busto(1989) Stroke 20:1627, Chen et al. (1996) Mol Endocrinol 10:682-693, andU.S. Pat. No. 6,017,965. According to this model, the middle cerebralartery is permanently occluded by passing a small piece of suture threadthrough the carotid artery to the region of the middle cerebral artery.Core body temperature is maintained at 37° C. This model is differentfrom the bilateral common carotid artery occlusion in gerbil ineliciting a more restricted brain infarct, and thereby approximating adifferent kind of stroke (focal thrombotic stroke).

Transient Focal Cerebral Ischemia. In another model of focal cerebralischemia in the rat, the middle cerebral artery is temporarily occludedby passing a small piece of suture thread through the carotid artery tothe region of the middle cerebral artery. The suture thread is withdrawnafter an ischemic period of 2 hours. Core body temperature is maintainedat 37° C.

Additional models of focal ischemia include, but are not limited to,photochemically induced focal cerebral thrombosis, blood clotembolization, microsphere embolization and related methods. See McAuley(1995) Cerebrovasc Brain Metab Review 7:153-180.

Renal Ischemia. Adult male rats are anesthetized with phenobarbital (50mg/kg) and the body temperature of rats is maintained between 36-37° C.Renal ischemia is induced by clamping the left renal artery for 15minutes (mild ischemia) or 45 minutes (severe ischemia), followed byreperfusion for 5 hours, as disclosed in Kuznetsov (1996) Proc Natl AcadSci USA 93:8584-8589.

XX.B. In vitro Models of Ischemia

Cell Culture Model of Epithelial Ischemia. Canine kidney (MDCK) cellsare grown in Dulbecco's minimal essential medium supplemented with 5%fetal bovine serum. Rat thyroid (PCC13) cells are grown in Coon'smodified Ham's F-12 medium (Sigma of St. Louis, Mo.) supplemented with5% bovine calf serum and a hormone mixture as described in Grollman etal. (1993) J Biol Chem 268:3604-3609. Cultured MDCK or PCC13 cells aresubjected to inhibition of oxidative metabolism by treatment withantimycin A, a specific inhibitor of mitochondrial oxidativephosphorylation as disclosed in Ramachandran & Gottlieb (1961) BiochimBiophys Acta 53:396-402. Alternatively, or in addition, the cells can betreated with 2-deoxyglucose, a nonhydrolyzble analog of glucose, toinhibit glycolytic metabolism. See Bacalloa et al. (1994) J Cell Sci107:3301-3313, Mandel et al. (1994) J Cell Sci 107:3315-224, andKuznetsov (1996) Proc Natl Acad Sci USA 93:8584-8589.

Cell Culture Model of Oxygen and Glucose Deprivation. Chinese hamsterovary (CHO) cells are grown in Ham's F-10 medium containing 15% newborncalf serum (GibcoBRL of Gaithersburg, Md.). Cells (5 ml) are seeded at adensity of 150,000 cells per ml to T25 flasks (Corning of Acton, Mass.)and are used for experiments in a subconfluent state approximately 48hours later. To achieve glucose deprivation, 15% serum is added to F-10medium prepared without glucose, resulting in a partially glucosedeficient broth. During incubation, cells use the remaining glucoseafter about 20 hours, as can be determined using a Sigma glucosecolorimetric assay kit. Glucose-deprived cells are harvested after anadditional 24 hours of incubation.

To achieve anoxia, cultures in fell medium (or in full medium containing50% additional glucose) were placed in a sealed Brewer jar (BaltimoreBiological Laboratory, Microbiology Systems of Baltimore, Md.) andanaerobiosis was initiated by using a hydrogen generator in a 4-7%carbon dioxide atmosphere as described previously by Anderson & Matovcik(1977) Science 197:1371-1374 and Seip & Evans (1980) J Clin Microbiol11:226-233. The oxygen concentration in the jar is decreased to <0.4% in100 minutes, and the concentration of oxygen at cell depth in anonagitated solution is calculated to be within 1% of the environmentalvalue within 30 minutes. Such a calculation can be made according to themethods described in Gerweck et al. (1979) Cancer Res 39:966-972. Theformation of water vapor from hydrogen and oxygen causes a brief (about15 minute) temperature increase to about 38.6° C. in the culture mediumsoon after initiation of anaerobiosis. This increase is insufficient toelicit a heat-shock response.

Anoxia can be verified using a methylene blue indicator solution. Thissolution becomes colorless (indicating the absence of oxygen) 5-6 hoursafter the initiation of anaerobiosis. A constant glucose concentration(1 g/L) can be maintained by changing the medium at 24 hours prior toand immediately prior to the initiation of anaerobiosis.

Cell Culture Model of Cerebral Ischemia. Isolated neurons can becultured on a monolayer comprising a growth-permissive substrate, suchas an immobilized monolayer of a purified, growth-promoting factor, sucha monolayer comprising collagen, fibronectin, of the L1 glycoprotein. Asan exemplary procedure, neurons (post-natal days 2-7) are dissociated bytrypsinization essentially as described, for example, in U.S. Pat. No.5,932,542. Neurons are added to a well coated with a growth-promotingfactor, followed by addition of either a single concentration orincreasing concentrations of the candidate composition. Neurons arecultured overnight (about 16 hours) at 37° C., and then neuriteoutgrowth is measured. Hypoxia/anoxia can be achieved as describedherein above. Neurite outgrowth of cells subjected to ischemicconditions and to which a candidate therapeutic composition wasadministered can then be compared to neurite outgrowth on control cellsalso subjected to ischemic conditions without administration of atherapeutic composition.

Cell Culture Model of Glutamate-induced Oxidative Toxicity inHippocampus. Glutamate is the major excitatory transmitter in the brain,and is proposed to play a role in epileptic pathogenesis and seizureactivity. Numerous in vivo models involving different kinds of seizuresand behavioral effects that are relevant for clinically distinct formsof epilepsy are known. In vitro models of glutamate-induced oxidativetoxicity are also known, an exemplary procedure described herein. Themouse hippocampal cell line (Davis & Maher (1994) Brain Res652(1):169-173) is maintained in Dulbecco's modified Eagles' medium(GibcoBRL of Gaithersburg, Md.) with 10% fetal bovine serum (AtlantaBiologicals of Atlanta, Ga.). HT22 cells are seeded onto 96-well platesat 20,000 cells per well and cultured overnight at 37° C. in normalgrowth medium. Glutamate-induced oxidative toxicity is elicited byadministration of 2-10 mM glutamate or NMDA. Further methods aredisclosed in Su et al. (1998) J Mol Cell Cardiol 30(3):587-598; Xiao etal. (1999) J Neurochem 72:95-101, and U.S. Pat. No. 6,017,965.

XX.C. Assays for Recovery Following Ischemia or Other Stress Conditions

The effects of therapeutic compositions disclosed herein, may beexamined to determine potential therapeutic strategies for mitigatingand/or reversing cellular damage in these animal models. Exemplary,although not limiting, measures to assess therapeutic efficacy asdisclosed herein below.

Neurological Assessment Assay. Neurological deficit and recovery can bemonitored using standardized scores that represent careful observationof consciousness, respiration, cranial nerve activity, spinal nerveactivity, and motor function, as disclosed in U.S. Pat. No. 5,571,840.Interobserver variability can be resolved by consultation of thedetailed description of each neurological function. Additional assays ofcognitive, sensory, and motor impairment are disclosed in U.S. Pat. No.6,017,965.

Infarct Size Assa. The efficacy of candidate compounds disclosed hereincan also be evaluated by determination of infarct size followingadministration of the composition to an animal subjected to ischemicconditions. At a selected timepoint(s) following initiation of ischemicconditions, such an animal is sacrificed and processed for routinehistology suitable for the tissue of interest and according to methodswell-known in the art Image processing software (e.g. Bio Scan OPTIMASof Edmonds, Washington) can be utilized to facilitate accuratecalculation of infarct volume.

Detection of Molecular Markers for Cell Degeneration. In anotherembodiment, damaged tissue can be identified in brain sections byimmunolabeling with antibodies that recognize antigens such as Alz-50,tau, A2B5, neurofilaments, neuron-specific enolase, and others that arecharacteristic of neurodegeneration as disclosed in U.S. Pat. No.6,046,381. Immunolabeled cells can be quantified using computer-aidedsemiquantitative analysis of confocal images.

Cell Viability Assay. When in vitro models of ischemia are employed,cell viability can be assessed by measuring cell ability to metabolize3-(4,5-dimethyldiazol-2-yl)-2,5-dipehnyltetrazolium bromide (MTT) asdescribed in Hansen et al. (1989) Electrophoresis 10:645-652. Briefly 10μl of MTT solution (5 mg/ml) is added to cell cultures is 96-well platesand the cells are maintained in normal growth medium for 4 hours at 37°C. Solubilization solution (100 μl; 50% dimethylformamide and 20% sodiumdodecyl sulfate, pH 4.8) is then added directly to each culture inindividual wells of the 96-well plate. After an overnight incubation atroom temperature, absorbance is measured.

Alternatively, cell viability can be assessed by measuring the releaseof lactate dehydrogenase, a cytoplasmic enzyme that is released fromdying cells as disclosed in Choi et al. (1987) J Neurosci 7:357 and U.S.Pat. No. 6,017,965.

Neuronal Growth Assays. A cell culture model of neural ischemia asdescribed herein above can be evaluated by visual examination of labeledneuronal processes, and quantitation of the length, density, anddynamicism of neuronal processes (e.g. dendrites and spines) asdisclosed in Horch et al. (1999) Neuron 23:353-364 and McAllister et al.(1997) Neuron 18:767-778.

In another embodiment, molecular markers can be used to evaluate neuritegrowth in fixed brain tissue section. For example, brain sectionsderived from an animal model of ischemia can labeled using antibodiesthat recognize MAP-2 (a marker of neuronal cell bodies and dendrites)and for synaptophysin (a marker of presynaptic terminals). Labeledsections can be viewed on a confocal microscope and documented usingcomputer-aided semiquantitative analysis of confocal images. The area ofthe neuropil occupied by MAP-2-immunolabeled dendrites or bysynaptophysin-immunolabeled terminals can be quantified and expressed asa percentage of the total image area. See Masliah et al. (1992) ExpNeurol 136:107-122 and Toggas et al. (1,994) Nature 367:188-193.

Additional methods for assaying neuronal growth are disclosed in Dohertyet al. (1995) Neuron 14:57-66, Schnell et al. (1990) Nature 343:269-272,U.S. Pat. Nos. 5,250,414 and 5,898,066, and International PCTPublication WO 99/61585.

XXI. Disorders of Protein Transport

Protein misfolding disorders are a common component of numerous geneticdisease states including, but not limited to, cystic fibrosis, familialhypercholesterolemia, retinitis pigmentosa and al-antitrypsinmisfolding. Compounds that modulate the activity of the Hsp90 family ofmolecular chaperones can thus be used in accordance with a therapeuticmethod of the present invention for reversing the protein foldingdefects that identify the disease state or for enhancing proteintransport from the endoplasmic reticulum of a cell. Thus, a compoundthat modulates the conformation of GRP94 can be used to treat a diseasestate resulting from defects in protein transport into or from theendoplasmic reticulum. Compounds that abrogate GRP94 activity can beused for the treatment of a disease state, such as cancer, wherein atherapeutic benefit can be provided by blocking the egress of proteins(e.g., growth factors) from the endoplasmic reticulum. conversely,compounds that promote GRP94 activity can be used to treat a diseasewherein a therapeutic benefit can be provided by enhancing proteinexport from the endoplasmic reticulum.

To assess misregulation of protein transport, a model system thatmeasures epidermal growth factor receptor (EGF-R) levels and/orintracellular localization can be employed (Supino-Rosin et al. (2000) JBiol Chem 275(29):21850-21855). For example, the benzoquinone ansamaycingeldanamycin targets two Hsp90 molecular chaperones (Hsp90 and GRP94)and by inhibiting their activities, blocks and promotes its subsequentproteolytic degradation. In response to geldanamycin treatment, EGF-R isunable to traffic to the plasma membrane and the cell becomes refractoryto stimulation by EGF.

Laboratory Examples

The following Laboratory Examples have been included to illustratepreferred modes of the invention. Certain aspects of the followingLaboratory Examples are described in terms of techniques and proceduresfound or contemplated by the present inventors to work well in thepractice of the invention. These Laboratory Examples are exemplifiedthrough the use of standard laboratory practices of the inventors. Inlight of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following LaboratoryExamples are intended to be exemplary only and that numerous changes,modifications and alterations can be employed without departing from thespirit and scope of the invention. It will be understood that variousdetails of the invention may be changed without departing from the scopeof the invention. Furthermore, the foregoing description is for thepurpose of illustration only, and not for the purpose of limitation—theinvention being defined by the claims.

EXAMPLES 1-8 Ligand-Mediated Activation of GRP94 Molecular ChaperoneActivity

The amino terminal domain of Hsp90 chaperones contains an adenosinenucleotide binding pocket that binds the Hsp90 inhibitors geldanamycinand radicicol. The following Examples 1-8 demonstrate that bis-ANS (1-1′bis(4-anilino-5-napthalenesulfonic acid)), an environment-sensitivefluorophore that interacts with nucleotide binding sites, binds to theadenosine nucleotide binding domain of GRP94 and activates its peptidebinding and molecular chaperone activities. Bis-ANS, like heat shock,elicits a tertiary conformational change in GRP94 which activates GRP94function and is inhibited by radicicol. Confirmation of the N-terminalnucleotide-binding domain as the bis-ANS binding site was obtained bysequencing of bis-ANS-labeled GRP94 protease digestion products. Thesedata identify a ligand-dependent, allosteric regulation of GRP94 andsuggest a model for ligand-mediated regulation of GRP94 function.

Materials and Methods for Examples 1-8

Materials. Fluorescent probes were obtained from Molecular Probes(Eugene, Oreg.). Bis-ANS concentration was determined by absorbance at385 nm (ε₃₈₅=16,790 cm⁻¹ M⁻¹ in water). Citrate synthase (E.C. 4.1.3.7)was purchased from Boehringer Mannheim (Mannheim, Germany). Radicicolwas obtained from Dr. Len Neckers, National Cancer Institute, Frederick,Md. Peptide VSV8 (RGYVYQGL—SEQ ID NO:7) was synthesized by theUniversity of North Carolina at Chapel Hill Peptide Synthesis Facility(Chapel Hill, N.C.). Na [¹²⁵I] was purchased from Amersham Pharmacia(Piscataway, N.J.). All other reagents were obtained from Sigma ChemicalCo. (St. Louis, Mo.) unless otherwise indicated. GRP94 was purified fromporcine pancreas as described by Wearsch & Nicchitta (1996b)Biochemistry 35:16760-16769. The concentration of GRP94 was determinedby absorbance at 280 nm (1 mg/ml=1.18A₂₈₀).

Fluorophore Binding Reactions. All binding reactions, with the exceptionof the indicated circular dichroism and citrate synthase aggregationexperiments, were conducted in buffer A (110 mM KOAc, 20 mM NaCl, 2 mMMg(OAc)₂, 25 mM K-HEPES pH 7.2, 100 μM CaCl₂). Fluorescent probe andradicicol stocks were prepared in dimethyl formamide at 5 mM finalconcentration. For all assays, control reactions at solvent dilutionsidentical to experimental conditions were performed to correct for anysolvent effects. Where indicated, GRP94 was heat shocked by incubationin a 50° C. water bath for 15 minutes followed by cooling to 37° C.

Fluorescence Measurements. Emission spectra were obtained in aFLUOROMAX™ spectrofluorometer (SPEX Industries Inc. of Edison, NewJersey) operating in photon counting mode. Spectra were recorded andprocessed with DM3000f operating software, version 2.1 (SPEX IndustriesInc. of Edison, N.J.). For emission scans, slit width was set at 1 nm.Excitation wavelengths were as follows: Prodan, 360 nm; ANS, 372 nm;bis-ANS, 393 nm; tryptophan, 295 nm. All spectra were backgroundcorrected.

Circular Dichroism Measurements. Far-UV CD spectrometry was performed onan AVIV Associates 62DS™ circular dichroism spectrometer (AVIVAssociates of Lakewood, N.J.). Samples were analyzed in a 1 mm pathlength quartz cuvette at 37° C. GRP94 samples (1 μM) were prepared instandard phosphate buffered saline solution as buffer A producedunacceptable dynode voltages in the relevant region of the spectrum.GRP94 was incubated with 10 □M bis-ANS for 2 hours at 37° C. prior toobtaining spectra. Spectra were recorded from 300 to 195 nm. Theα-helical content of GRP94 was calculated from the molar ellipticity at222 nm. See Myers & Jakoby (1975) J Biol Chem 250:3785-3789.

Conformational Analysis by Proteolysis. The conformational state ofGRP94 was assessed by tryptic digestion of the protein and subsequentSDS-PAGE analysis. For simple proteolysis experiments, 10 μl of a 0.5mg/ml GRP94 stock, with or without prior heat shock, was combined with 1μl bis-ANS and/or radicicol stock solutions and incubated for theindicated times at 37° C. Samples were then combined with 0.1% trypsinand digested for 30 minutes at 37° C. An equal volume of SDS-PAGE samplebuffer was added and the samples were snap frozen in liquid nitrogen.Immediately prior to gel analysis, samples were thawed and boiled for 5minutes. Samples were then separated on 12.5% SDS-polyacrylamide gels.Gels were fixed and stained with Coomassie Blue. For time courseexperiments, excess free bis-ANS was removed immediately prior totrypsinization by gel filtration on 0.5 ml G-25 SEPHADEX® spin columns.

Identification of the bis-ANS binding site. The bis-ANS binding regionof GRP94 was identified by covalent incorporation of bis-ANS into GRP94following bis-ANS photolysis procedures described by Sharma et al.(1998) J Biol Chem 273(25):15474-78 and Seale et al. (1998) MethodsEnzymol 290:318-323. Briefly, 50 μg of GRP94 was combined with 50□Mbis-ANS in a final volume of 100 μl and photo-crosslinked for 15 minuteson ice with a 366 nm hand-held UV lamp (Ultra-violet Products, Inc. ofSan Gabriel, Calif.). Following photocrosslinking, GRP94-bis-ANScomplexes were digested with trypsin for one hour at 37° C. Thetrypsin-derived limit digestion products were then separated by C-18reverse phase HPLC using a continuous acetonitrile/water gradient in 20mM ammonium bicarbonate, with sequential detection by UV absorbance (220nm) and fluorescence emission (excitation 418 nm; emission 498 nm). Themajor resultant fluorescent peak was collected and the correspondingpeptide sequenced by Edman degradation on an Applied Biosystems PROCISE™model 492 automated protein sequencer.

Native Blue Electrophoresis. The oligomeric state of GRP94 was assayedby blue native polyacrylamide gel electrophoresis (BN-PAGE) as describedby Schagger et al. (1994) Anal Biochem 217:220-230. GRP94 was eitherheat shocked or exposed to a 10-fold molar excess of bis-ANS for theindicated times. Samples were then dissolved in 15% glycerol and loadedonto 5-18% gradient gels with 0.02% Coomassie Brilliant Blue in thecathode buffer. Gels were run at 4° C., stained with Coomassie Blue,de-stained and dried.

Citrate Synthase Aggregation Assays. The effects of GRP94 on the thermalaggregation of citrate synthase were assayed by the methods described byBuchner et al. (1998) Methods Enzymol 290:323-338. Samples containing noprotein, or GRP94 (1 μM), were incubated in 40 mM HEPES pH 7.5 for twohours at 37° C. with either 0.2% DMF or 10 □M bis-ANS. The samples werethen warmed to 43° C. for five minutes and placed in aspectrofluorometer thermostatted at 43° C. Citrate synthase was thenadded to 0.15 μM final concentration and the thermal aggregation of theenzyme followed by light scattering. Excitation and emission wavelengthswere both 500 nm with 2 nm slit width. The time course of citratesynthase aggregation was followed for 1000 seconds.

Peptide Binding to GRP94. Iodination of VSV8 was performed by theIODOBEADS™ procedure (Pierce Chemical Co. of Chicago, Ill.), andunincorporated [¹²⁵I] was removed by fractionation on a SEP-PAK™ C18reverse-phase cartridge. Iodinated peptide was mixed with unlabeledpeptide to yield a final specific activity of 6.0 □Ci/mg. GRP94 (4.7 μg,final concentration 0.5 μM) was incubated with an equimolar quantity ofbis-ANS in 0.1% DMF in 100 μL buffer A for 3.5 hr at 37° C. Samples werethen incubated for an additional 30 min at 37° C., or heat shocked for15 min at 50° C. and allowed to recover for 15 min at 37° C. A ten-foldmolar excess of [¹²⁵I ]VSV8 was added (final concentration 5 μM) and themixture incubated for 30 min at 37° C. All incubations were performed inthe dark to prevent bis-ANS degradation. Samples were then eluted on1.2-mL SEPHADEX® G-75 spin columns pre-blocked with 75 μg BSA, and[¹²⁵I] was quantitated by gamma counting.

Example 1 Binding of Polarity-sensitive Fluorescent Probes to GRP94

Recent studies on the conformational regulation of GRP94 have identifieda tertiary structural change that occurs in response to heat shock andis associated with an activation of peptide binding activity. SeeWearsch et al. (1998) Biochemistry 37(16):5709-16, Sastry & Linderoth(1999) J Biol Chem 274:12023-12035. Coincident with the heatshock-elicited conformational change, GRP94 displays enhanced binding ofenvironment sensitive fluorescent probes such as Nile Red, whichpreferentially bind to hydrophobic domains (Wearsch et al., 1998). GRP94contains two domains of significant hydrophobicity, a C-terminalassembly domain and a highly conserved N-terminal region, whichcorresponds to the Hsp90 geldanamycin and adenosine nucleotide bindingsite. See Stebbins et al. (1997) Cell 89:239-250; and Prodromou et al.(1997) Cell 90:65-75.

To characterize the structural basis for the heat shock dependentactivation of GRP94 activity, the interaction of polarity-sensitivefluorophores with native and heat shocked GRP94 was examined. The threeprobes tested, Prodan (6-propionyl-2-(dimethylamino)naphthalene), 8-ANS(1,8-anilinonaphthalenesulfonate) and bis-ANS(bis(1,8-anilino-naphthalenesulfonate) are structurally related probesthat bind to hydrophobic sites on proteins and undergo substantialfluorescence spectrum changes upon introduction into nonpolarenvironments, as discussed by Rosen & Weber (1969) Biochemistry8:3915-3920; Weber & Farris (1979) Biochemistry 18:3075-3078; Takashi etal. (1977) Proc Natl Acad Sci USA 74:2334-2338; Shi et al. (1994)Biochemistry 33:7536-7546. The following experimental protocol wasutilized. GRP94 was warmed to 37° C. and either maintained at 37° C. orheat shocked for 15 minutes at 50° C., followed by incubation at 37° C.Subsequently, probe stocks were added to the GRP94 stocks and emissionspectra recorded after 30 min at 37° C.

As depicted in FIG. 1A, the emission maxima of Prodan in the presence ofnative or heat shocked GRP94 were essentially identical, indicating thatProdan does not interact with the hydrophobic binding pocket(s)displayed by heat shocked GRP94. In contrast, the structurally relatedprobe, 8-ANS, displays weak interactions with native GRP94, yet bindsavidly following heat shock (FIG. 1B).

The interaction of bis-ANS with GRP94 was complex, and displayed a cleartime dependence. As depicted in FIGS. 1C and 1D, the initial bis-ANSbinding to native GRP94 was bi-phasic and following extended incubationsin the presence of bis-ANS, a level of fluorophore binding similar tothat seen with heat shocked GRP94 was observed. These data suggest thatmaximal bis-ANS binding to GRP94 required a slow structural transition.This transition further suggests a bis-ANS elicited conformationalchange in GRP94 and/or the bis-ANS dependent stabilization of aconformation state accessed at low frequency by the native protein.

Example 2 Analysis of bis-ANS Binding to Heat Shocked GRP94

To determine the affinity of bis-ANS for GRP94, bis-ANS was added toincreasing concentrations of heat shocked GRP94, the fluorescencespectrum was determined, and the emission intensity at 475 nm plotted asa function of GRP94 concentration (FIGS. 2A and 2B). Under theexperimental conditions used in this series of experiments, bis-ANSbinding to GRP94 was near maximal at a 20-fold molar excess of GRP94monomer over bis-ANS, with half maximal binding observed at 110 nM GRP94(FIG. 2B). Importantly, these data indicate that bis-ANS binds in asaturable manner to heat shocked GRP94 and that the site(s) of bis-ANSbinding to GRP94 displayed similar relative affinities for bis-ANS.

Example 3 Structural Consequences of bis-ANS Binding to GRP94

Following an extended incubation period, the emission spectra of bis-ANSbound to native GRP94 bears substantial similarity to that emissionspectra of bis-ANS bound to heat shocked GRP94. Because heat shock isknown to elicit a stable tertiary conformational change in GRP94(Wearsch et al. (1998) Biochemistry 37(16):5709-16) these data suggestthat the binding of bis-ANS to GRP94 induces, or stabilizes, aconformational change similar to that occurring in response to heatshock. To determine whether the GRP94 conformation seen upon addition ofbis-ANS is similar to that observed following heat shock, a series ofstructural studies on the bis-ANS/GRP94 complex was performed.

In one series of experiments, the proteolysis patterns of native, heatshocked and bis-ANS treated GRP94 were examined. As shown in FIG. 3A,lanes 2 and 3, incubation of native GRP94 with low levels of trypsinyields two prominent proteolysis products, representing known structuraldomains of the protein, as described by Stebbins et al. (1997);Prodromou et al. (1997) Cell 90:65-75; Wearsch & Nicchitta (1996b)Biochemistry 35:16760-16769. In contrast, proteolysis of either bis-ANStreated or heat shocked GRP94 yields a substantially reduced recovery ofthe prominent proteolysis products, with the concomitant appearance of adiverse array of proteolytic fragments of higher SDS-PAGE mobility.Essentially identical proteolysis patterns were observed followingeither heat shock or bis-ANS treatment of HSP90.

These data provide evidence that bis-ANS binding to GRP94 elicits orstabilizes GRP94 in a conformation similar to that occurring in responseto heat shock, suggesting that there exists a GRP94 conformation statethat can be readily accessed and/or stabilized by either heat shock orligand (bis-ANS) binding.

Example 4 Effects of bis-ANS Binding on GRP94 Quaternary and SecondaryStructure

When purified from tissue, GRP94 exists as a homodimer, as described byWearsch & Nicchitta (1996a) Prot Express Purif 7(1):114-21; Nemoto etal. (1996) J Biochem 120:249-256. Following heat shock however, GRP94forms higher molecular weight complexes, as described by Wearsch et al.(1998) Biochemistry 37:5709-5719. To further characterize the effects ofbis-ANS on GRP94 structure, the oligomerization states of native, heatshocked and bis-ANS treated GRP94 were assayed by the blue nativepolyacrylamide gel electrophoresis (BN-PAGE) technique described bySchagger et al. (1994). In these experiments, GRP94 was incubated withbis-ANS or briefly heat shocked and subsequently incubated at 37° C. Thesamples were then analyzed by BN-PAGE. As seen in FIG. 4, in the absenceof heat shock or bis-ANS treatment the majority of GRP94 exists as adimer with an apparent molecular weight of approximately 200 kDa.However, exposure to heat shock causes a relatively rapid formation oftetramers, hexamers, and octamers (FIG. 4, lanes 2-4). Incubation ofGRP94 with a ten-fold molar excess of bis-ANS induces changes in thequaternary structure of GRP94 that mimic those seen upon heat shock(FIG. 4, lanes 4, 5). These data lend further support to the hypothesisthat bis-ANS induces or stabilizes a structural transition in GRP94 thatis similar to that occurring in response to heat shock.

To gain further insight into the nature of the bis-ANS dependentconformational change, GRP94 was subjected to heat shocked or treatedwith bis-ANS and far-UV CD spectra obtained (FIG. 5). As shown in FIG.5, the CD spectra for native, heat shocked, and bis-ANS treated GRP94were identical, indicating that bis-ANS binding does not alter GRP94secondary structure.

Example 5 Radicicol Inhibits Temperature and bis-ANS Induced GRP94Conformational Changes

Radicicol, a macrocyclic antibiotic, binds to the highly conservedN-terminal nucleotide binding pocket of HSP90 and thereby blocks HSP90function. (Sharma et al. (1998) Oncogene 16(20):2639-45; Roe et al.(1999) J Med Chem 42:260-266). To determine if radicicol binding alsoinfluenced the structural dynamics of GRP94, the following experimentswere performed. GRP94 was incubated with increasing concentrations ofradicicol, heat shocked, cooled, and digested with trypsin. SubsequentSDS-PAGE analysis of the samples showed that in the presence ofradicicol, GRP94 was unable to undergo the heat shock-induced structuraltransition, as assayed by the similarities in proteolysis patternsbetween native GRP94 and radicicol-treated, heat shocked GRP94. Similarinhibition of the heat shock induced structural transition of HSP90 byradicicol was also observed.

To determine if radicicol could also inhibit the bis-ANS dependent GRP94structural transition, GRP94 was incubated with increasingconcentrations of radicicol, bis-ANS was then added, and the sampleswere incubated for one hour. Samples were subsequently digested withtrypsin and the proteolysis patterns determined by SDS-PAGE. As isdepicted in FIG. 6A, radicicol, when present at a ten-fold molar excessover bis-ANS, efficiently blocked the bis-ANS-dependent GRP94conformation change.

Though the experiment depicted in FIG. 6A indicated that radicicol wasable to inhibit the appearance of the bis-ANS-dependent conformationalstate, it was necessary to determine if bis-ANS binding to GRP94 wasblocked by radicicol treatment. To this end, the following experimentwas performed. GRP94 was incubated in the presence of increasingconcentrations of radicicol, subsequently heat treated under conditionssufficient to elicit efficient bis-ANS binding, and bis-ANS bindingassayed. As shown in FIG. 6B, radicicol, in a dose-dependent manner,inhibited bis-ANS binding to heat-treated GRP94.

Because radicicol itself blocks the heat shock-induced conformationchange, these data present two models of bis-ANS action. In one model,bis-ANS binds to the nucleotide binding domain and directly elicits theobserved conformational change. Radicicol, by binding to the adenosinenucleotide binding pocket, would then be predicted to inhibit thebis-ANS-dependent conformational change. In an alternative model, GRP94interconverts, in a temperature sensitive manner, between twoconformational states, arbitrarily referred to as the open or the closedstate. In the open state, bis-ANS bind and thereby stabilizes the openconformation whereas radicicol binding would stabilize the closedconformation. For both models, bis-ANS binding to the N-terminaladenosine nucleotide binding domain was predicted and was subsequentlyexamined.

Example 6 bis-ANS Binds to the N-terminal AdenosineNucleotide/Radicicol/Geldanamycin Binding Domain

Having determined that bis-ANS can alter the conformation of GRP94, thesite of bis-ANS binding to GRP94 was targeted for identification.Irradiation of bis-ANS with UV light allows the covalent incorporationof the probe into protein binding sites, as described by Sharma et al.(1998) J Biol Chem 273(25):15474-78 and Seale et al. (1998) MethodsEnzymol 290:318-323. As described in Materials and Methods, GRP94 wascombined with an excess of bis-ANS and photo-crosslinked on ice for 15minutes. GRP94 was subsequently digested with trypsin, the fluorescentpeptides purified by HPLC, and the sequence of the labeled peptidesdetermined by Edman sequencing. The major resultant fluorescent peptideyielded the sequence YSQFINFPIYV (SEQ ID NO:8), which mapped to residues271-281 of the N-terminal domain of GRP94. This segment is homologous tothe human HSP90 sequence HSQFIGYPITLFV (SEQ ID NO:9) from amino acids210-222, and overlaps with the C-terminal region of the adenosinenucleotide/geldanamycin/radicicol binding domain (Stebbins et al. (1997)Cell 89:239-250; Prodromou et al. (1997) Cell 90:65-75).

Example 7 Bis-ANS Activates GRP94 Chaperone Activity

To determine if the bis-ANS-dependent conformational changes in GRP94were of functional significance, the molecular chaperone activities ofnative, heat shocked and bis-ANS treated GRP94 were evaluated in athermal aggregation assay, as described by Jakob et al. (1995) J BiolChem 270:7288-7294 and Buchner et al. (1998) Methods Enzymol290:323-338. In these experiments, citrate synthase aggregation wasassayed in the presence of buffer, native GRP94, heat shocked GRP94 orGRP94 that had been previously exposed to bis-ANS for two hours.Following experimental treatment of the GRP94, reactions wereequilibrated at 43° C., citrate synthase then added and aggregation, asrepresented by light scattering, was measured.

In the absence of GRP94, citrate synthase undergoes rapid thermalaggregation and under the experimental conditions depicted in FIG. 7A,reaches a plateau level within 15 min. In the presence of native GRP94,the degree of aggregation is reduced, suggesting that at least afraction of the population of native GRP94 molecules are in an activeconformation. Under these experimental conditions, approximately 50% ofthe citrate synthase aggregated. At the concentration of GRP94 used inthese experiments, and assuming a stoichiometric interaction, theseresults indicate that roughly 8% of the native GRP94 is in the activeconformation. In the presence of heat shocked or bis-ANS treated GRP94,no thermal aggregation of citrate synthase was detectable (FIG. 7A).These data indicate that the ability of GRP94 to bind to substrateproteins is enhanced by prior heat shock or bis-ANS treatment andsuggest that the GRP94 conformation elicited by heat shock or bis-ANSbinding represents an active state of the molecule.

Example 8 bis-ANS Activates Peptide Binding Activity to GRP94

To assess the effects of bis-ANS treatment on the peptide bindingactivity of GRP94, GRP94 was either treated with bis-ANS, or brieflyheat shocked. A ten-fold molar excess of [¹²⁵I]-VSV8 was then added andthe mixture incubated for 30 min at 37° C. Free peptide was separatedfrom bound peptide by SEPHADEX® G75 spin column chromatography and thebound peptide was quantitated by gamma counting. As shown in FIG. 7B,treatment of GRP94 with bis-ANS significantly enhanced the peptidebinding activity of GRP94, yielding approximately a four to five-foldstimulation over native protein. Under similar conditions, heat shockedGRP94 displayed approximately a ten-fold stimulation of binding. Fromthe data presented in FIGS. 7A and 7B, it is apparent that bis-ANSelicits or stabilizes a GRP94 conformation that displays markedlyenhanced molecular chaperone and peptide binding activities.

SUMMARY OF EXAMPLES 1-8

Examples 1-8 demonstrate that bis-ANS binds to the conserved, N-terminaladenosine nucleotide binding domain of GRP94 and elicits a tertiaryconformational change yielding markedly enhanced molecular chaperone andpeptide binding activities. The binding of bis-ANS to GRP94 isbi-phasic, with an initial rapid binding phase followed by a slow,extended binding phase. In accord with these data, bis-ANS binds to andstabilizes a low abundance GRP94 conformation, referred to as the openstate. In this model, GRP94 molecular chaperone and peptide bindingactivity is intimately coupled to such a conformation change. While itis not applicants' desire to be bound by any particularly theory or act,in the absence of regulatory ligands, access to this conformation isbelieved to occur in a time and temperature-dependent manner throughintrinsic structural fluctuations. Inhibitory ligands, such asgeldanamycin and radicicol, function by binding to and stabilizing GRP94in a closed, or inactive, conformation.

Summarily, Examples 1-8 disclose the identification of a ligand elicitedconformational change in GRP94 that is accompanied by a markedactivation of molecular chaperone and peptide binding activities. Thesimilarities between the conformations of GRP94 following heat shockactivation and bis-ANS binding support the conclusion that GRP94conformation and activity can be regulated by ligand binding to theN-terminal adenosine nucleotide binding domain and that the conformationof the protein in the bis-ANS liganded state is physiologicallyrelevant.

EXAMPLES 9-13 Allosteric Ligand Interactions in the Adenosine NucleotideBinding Domain of the Hsp90 Chaperone, GRP94

Examples 9-13 disclose that GRP94 and HSP90 differ in their interactionswith adenosine-based ligands. GRP94 displayed high affinity saturablebinding of the adenosine derivative N-ethylcarboxamido-adenosine (NECA),whereas HSP90 did not. In NECA displacement assays, GRP94 exhibited weakbinding affinities for ATP, ADP, AMP, adenosine and cAMP. GRP94 ATPaseactivity, though present, was non-saturable with respect to ATPconcentration and thus could not be characterized by traditionalenzymatic criteria. Radioligand and calorimetric studies of NECA bindingto GRP94 revealed that ligand binding to the nucleotide binding domainis under allosteric regulation. GRP94 is thus regulated through aligand-based allosteric mechanism and through regulatory adenosine-basedligand(s) other than ATP.

Materials and Methods for Examples 9-13

Purification of GRP94, BiP and Hsp90. GRP94 was purified from porcinepancreas rough microsomes as described by Wearsch & Nicchitta (1996a)Prot Express Purif 7:114-121 with the following modifications. Roughmicrosomes were washed after the initial isolation by 10-fold dilutionin 0.25M sucrose, 20 mM KOAc, 25 mM K-Hepes, pH 7.2, 5 mM Mg(OAc)₂ andsubsequent re-isolation by centrifugation (30 min, 40K rpm, 4° C.,Ti50.2 rotor). To release the lumenal contents from the isolated roughmicrosomes, the microsomes were permeabilized by addition of 5 mM CHAPSand the lumenal contents were subsequently isolated by centrifugationfor 2 hours at 45,000 RPM (4° C., Ti50.2 rotor).

BiP was purified by the following procedure. A lumenal protein fractionobtained from porcine pancreas rough microsomes was cycled overnightthrough a 1 ml ADP-agarose and a 1 ml ATP-agarose (Sigma Chemical Co. ofSt. Louis, Mo.) column coupled in series. The columns were then washedwith 2×5 ml of a buffer containing 350 mM NaCl, 25 mM Tris, pH 7.8, 5 mMMg²⁺ and the BiP was eluted from the nucleotide affinity columns with3×5 ml of the identical buffer supplemented with 10 mM ATP and ADP. TheBiP containing fractions were identified by SDS-PAGE, and dialyzedagainst 2×4 L of buffer A (110 mM KOAc, 20 mM NaCl, 25 mM K-Hepes, pH7.2, 2 mM Mg(OAc)₂ 0.1 mM CaCl₂). The protein sample was then applied toa SUPERDEX® 26/60 column (Amersham Pharmacia Biotech of Piscataway,N.J.) equilibrated in buffer A, and the BiP containing fractions, againidentified by SDS-PAGE, were pooled and concentrated by centrifugalultrafiltration (CENTRICON-30®; Amicon of Beverly, Mass.).

Hsp90 was purified from rat liver cytosol as follows. Cytosol wasadjusted to 30% ammonium sulfate and stirred for 60 min on ice. Thesolution was centrifuged at 20,000×g in a Sorvall SS34 rotor for 15minutes and the supernatant collected and filtered through a 0.22 μmfilter. The filtered supernatant was supplemented with proteaseinhibitors (1 μg/ml pepstatin, 1 μg/ml leupeptin, 20 μg/ml SBTI, and 0.5mM PMSF) and loaded onto a phenyl-SUPEROSE™ HR10/10 column (AmershamPharmacia Biotech of Piscataway, N.J.). After washing, the boundproteins were eluted with a gradient of 30-0% saturated ammonium sulfatein 10 mM Tris/HCl, pH 7.5, 1 mM EGTA, 0.5 mM DTT and the Hsp90containing fractions were identified by SDS-PAGE. The Hsp90 containingfractions were then pooled and dialyzed 2×3 hr against 2 L of low saltbuffer (10 mM NaCl, 25 mM Tris, pH 7.8). The dialyzed sample was thenfiltered through a 0.22μm filter, and injected onto a MONO-Q™ HR 10/10column (Amersham Pharmacia Biotech of Piscataway, N.J.) equilibrated inlow salt buffer. The column was eluted with a gradient of 10 mM-750 mMNaCl in 25 mM Tris, pH 7.8. The Hsp9O-containing fractions wereidentified by SDS-PAGE and pooled.

Further purification was achieved by applying the MONO-Q™ pool to a 4 mLhydroxylapatite column (Bio-Rad HTP of Hercules, Calif.) equilibrated inbuffer B (10 mM NaH₂PO₄, pH 6.8, 10 mM KCl and 90 mM NaCl). Thehydroxylapatite column was eluted with a 10 mM NaH₂PO₄ to 250 mMNaH₂PO₄, gradient and the Hsp90 fractions were identified by SDS-PAGE.The Hsp90 pool, in 225 mM NaH₂PO₄, 10 mM KCl, and 90 mM NaCl, wasconcentrated by centrifugal ultrafiltration (CENTRICON®-30; Amicon,Beverly, Mass.) and stored at −80° C.

[³H]-NECA Binding Assay. Nine μg of GRP94 was incubated with 20 nM[³H]-NECA (Amersham Pharmacia Biotech of Piscataway, N.J.), and variousconcentrations of competitors for one hour on ice in a final volume of250 μl of 50 mM Tris, pH 7.5. Where indicated, binding reactions wereperformed in either buffer C (10 mM Tris, pH 7.5, 50 mM KCl, 5 mM MgCl₂,2 mM DTT, 0.01% NP-40, 20 mM Na₂MoO₄) or 50 mM Tris, pH 7.5, 10 mMMg(OAc)₂. Bound versus free [³H]-NECA was assayed by vacuum filtrationof the binding reactions on #32 glass fiber filters (Schleicher andSchuell of Keene, New Hampshire), pre-treated with 0.3%polyethyleneimine (Sigma Chemical Co. of St. Louis, Mo.). Vacuumfiltration was performed with an Amersham Pharmacia Biotech (Piscataway,N.J.) vacuum filtration manifold.

Filters were rapidly washed with 3×4 ml of ice cold 50 mM Tris, pH 7.5,placed in 5 ml of scintillation fluid (SAFETYSOLVE™, RPI of Mt.Prospect, Ill.), vortexed, and counted by liquid scintillationspectrometry. In experiments in which the kinetic parameters of[³H]-NECA binding to GRP94 were determined, the chemical concentrationand specific activity of NECA was adjusted by addition of unlabeledNECA. All binding reactions were performed in triplicate and correctedby subtraction of background values, determined in binding reactionslacking GRP94.

ATP Binding Assay. Six μg of GRP94, BiP, and Hsp90 was incubated with 50μM γZ[³²P] ATP (1000 μCi/μmol) (Amersham Pharmacia Biotech ofPiscataway, N.J.) in buffer B on ice for 1 hour. Nitrocellulose filters(BA85) (Schleicher & Schuell of Keene, N.H.) were individually wet inbuffer B before use, and bound versus free [³²P]-ATP was separated byvacuum filtration. Filters were washed with 3×2 mL of ice cold buffer B,placed in 5 mL of scintillation fluid, vortexed, and counted.

Isothermal Titration Calorimetry. Isothermal calorimetry experimentswere performed at 25° C. using a MSC calorimeter (MicroCal Inc. ofNorthampton, Mass.). To determine the NECA binding parameters, two 5 μlinjections were followed by twenty-three 10 μL injections from a 152 μMNECA stock. The reaction chamber (1.3 mL) contained 5 μM GRP94.Necessary corrections were made by subtracting the heats of dilutionresulting from buffer addition to protein solution and ligand solutioninto buffer. The corrected data were then fit by the ORIGIN™ software(Microcal Software, 1998) to obtain the binding parameters. Theradicicol binding parameters were obtained in a similar manner with 5 μMGRP94 and 115 μM radicicol.

Phosphorylation Assays. To assay for GRP94 autophosphorylation, 1 μMGRP94 was incubated with γ-[³²P]ATP (6000 cpm/pmol) (Amersham PharmaciaBiotech of Piscataway, N.J.), diluted with cold ATP to yield a finalconcentration of 0.15 mM ATP in a buffer containing 10 mM Mg(OAc)₂ and50 mM K-Hepes, pH 7.4. For the casein kinase assay, 1 unit of caseinkinase II was incubated as described above, with the addition of 4 μMcasein. Competitors were added to the appropriate samples to yield finalconcentrations of 180 μM NECA in 3.6% DMSO, 180 μM radicicol in 3.6%DMSO, 5 μg/ml heparin, 5 mM GTP, or 3.6% DMSO. The 25 μl reactionmixtures were incubated at 37° C. for 1 hour and quenched by addition of10% trichloroacetic acid. Samples were analyzed by 10% SDS-PAGE gels andthe phosphorylated species were quantitated using a Fuji MACBAS1000™phosphorimaging system (Fuji Medical Systems of Stamford, Conn.).

ATPase Assay. 100 μl reactions consisting of 1 μM GRP94 monomer, variousconcentrations of MgATP (pH 7.0), and 50 mM K-Hepes, pH 7.4, wereincubated for two hours at 37° C. Samples were then spun through aCENTRICON®-30 (Amicon of Beverly, Mass.) at 10,000 rpm, 4° C. toseparate protein from nucleotide. A final concentration of 50 mM(NH₄)₂HPO₄, pH 7.0, and 4 μM AMP, pH 7.0, was added to dilutions of theabove samples and centrifuged at 15,200 rpm for 5 minutes at 4° C. 100μL of supernatant was then fractionated on a PARTISIL™ SAX column(Alltech of Deerfield, Ill.), using a Series 1050 Hewlett Packard HPLCsystem. Elution of nucleotides was performed by step gradient elutionusing a mobile phase of 150 mM (NH₄)₂HPO₄, pH 5.2, at 1.2 ml/min for thefirst ten minutes, followed by 300 mM (NH₄)₂HPO₄, pH 5.2, at a flow rateof 2 ml/min for the remainder of the elution. In this protocol, ADP andATP were well resolved, with ADP eluting at 7 minutes and ATP at 12minutes. Peak height values were used in calculations of percenthydrolysis and ADP formation. Spontaneous hydrolysis was determined foreach ATP concentration in paired incubations lacking GRP94. The AMP wasused as an internal reference standard to control for equivalent sampleloading.

Tryptophan Fluorescence. Tryptophan fluorescence measurements wereconducted in a FLUOROMAX™ spectrofluorometer (Spex Industries, Inc. ofEdison, N.J.) with the slit widths set to 1 nm for both excitation andemission. Samples were excited at a wavelength of 295 nm and theemission spectra were recorded from 300-400 nm. All spectra werecorrected by subtraction of buffer or buffer plus ligand samples. GRP94(50 μg/ml) was incubated in buffer A supplemented with 10 mM Mg(OAc)₂and the following concentrations of ligands for 1 hour at 37° C. (50 μMNECA, 50 μM geldanamycin, 2.5 mM ATP, or 2.5 mM ADP). Samples were thencooled to room temperature, transferred to a quartz cuvette, and thespectra collected. In control experiments, free tryptophan fluorescencewas not significantly influenced by the presence of any of the assayedligands.

Example 9 Hsp90 Proteins Differ in Adenosine-based Ligand BindingProperties

To determine whether Hsp90 and GRP94 displayed distinct adenosine-ligandbinding properties, the relative NECA and ATP binding activities ofGRP94, Hsp90 and BiP, the endoplasmic reticulum Hsp70 paralog, werecompared (FIG. 8). In these assays, purified GRP94, Hsp90 or BiP wereincubated on ice for 60 min in the presence of 20 nM [³H]-NECA and thebound versus free NECA resolved by vacuum filtration. As is evident inFIG. 8, whereas GRP94 displayed readily detectable [³H]-NECA bindingactivity, [³H]-NECA binding was not observed for Hsp90 or BiP. Insimilar experiments, [³H]-NECA binding to Hsp90 was evaluated in thepresence of molybdate and NP-40, which are known to stabilize the Hsp90conformation associated with ATP binding, as described by Sullivan etal. (1997). Under these conditions, [³H]-NECA binding to Hsp90 was againnot observed.

When ATP binding was assayed, BiP displayed the expected ATP bindingactivity whereas no ATP binding was observed to Hsp90 or GRP94. Asdiscussed below, the inability to detect ATP binding to Hsp90 is likelya consequence of the low affinity of Hsp90 for ATP (Prodromou et al.(1997) Cell 90:65-75; Scheibel et al. (1997) J Biol Chem272:18608-18613). In summary, these data indicate that GRP94 and Hsp90differ in their ability to bind the adenosine-based ligand NECA, andsuggest that the ligand specificity of the adenosine nucleotide bindingpocket of GRP94 differs from that of Hsp90.

Example 10 Kinetic Analysis of NECA Binding to GRP94

A kinetic analysis of [³H]-NECA binding to mammalian GRP94 is depictedin FIGS. 9A and 9B. [³H]-NECA binding to GRP94 was saturable, with a Kdof 200 nM and displayed a binding stoichiometry of 0.5 mol [³H]-NECA/molGRP94 monomer. These values are similar to those observed with placentalGRP94 (adenotin) by Hutchison et al. (1990) Biochemistry 29:5138-5144. AHill plot of the binding data yielded a slope of 1.2, indicating that[³H]-NECA binding to GRP94 was not cooperative.

Structurally, GRP94 exists as a dimer of identical subunits as describedby Wearsch & Nicchitta (1996a) Prot Express Purif 7:114-121; Wearsch &Nicchitta (1996b) Biochemistry 35:16760-16769; Nemoto et al. (1996) JBiochem 120:249-256). Given that the two subunits are identical, a 50%ligand occupancy at binding saturation was unexpected. The dissociationrate of NECA from GRP94 is rapid (Huttemann et al. (1984) NaunynSchmiedebergs Arch Pharmacol. 325:226-33) and so it was considered thatthe observed fractional occupancy level could reflect an artifact of themethod used to separate bound vs. free [³H]-NECA.

To evaluate the accuracy of the half-site occupancy value, the kineticsof NECA-GRP94 interaction were evaluated by isothermal titrationcalorimetry, a method that does not require the physical separation ofbound and free ligand. In these experiments, illustrated in FIG. 9C, thebinding stoichiometries of GRP94 for NECA and radicicol were determined.Radicicol is an antibiotic inhibitor of Hsp90 function that binds to theN-terminal nucleotide binding pocket of Hsp90 with high affinity (19 nM)and the expected binding stoichiometry of 2 mol radicicol/mol Hsp90dimer, as proposed by Roe et al. (1999) J Med Chem 42:260-266. Analysisof NECA binding to GRP94 by isothermal titration calorimetry yielded abinding stoichiometry of 1.1 mol NECA/mol GRP94 dimer. (FIG. 9C).

Radicicol, in contrast, bound at a stoichiometry of 2 mol radicicol/molGRP94 dimer, as shown in FIG. 9C. These data indicate that whileradicicol can achieve full occupancy of the two nucleotide binding sitespresent in the native GRP94 dimer, other ligands, such as NECA, eitherbind to a single unique site on GRP94, or upon binding to one of thenucleotide binding sites, elicit a conformational change in the pairedsite that prevents further ligand binding.

Example 11 Specificity of Ligand Binding to the Nucleotide BindingPocket of GRP94

To determine whether NECA bound to a single unique site on GRP94 or,alternatively, displayed half-site occupancy of the N-terminal adenosinenucleotide binding pockets, experiments were first performed todetermine if NECA binds to the adenosine nucleotide binding pocket.[³H]-NECA competition assays were performed with geldanamycin andradicicol, both of which are known to bind with high affinities to thenucleotide binding pocket of Hsp90 (Roe et al. (1999) J Med Chem42:260-266, Lawson et al. (1998) J Cell Physiol 174:170-8). The datadepicted in FIG. 10A indicate that both geldanamycin and radicicolcompete with [³H]-NECA for binding to GRP94 and do so with high relativeaffinities and in the following rank order, radicicol>geldanamycin.

As described Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156, it isdifficult to detect stable binding of ATP to GRP94. Should GRP94 displaya similar and quite low affinity for ATP, as reported for Hsp90 (Kd=132μM) by Prodromou et al. (1997) Cell 90:65-75, it would be very unlikelythat ATP binding could be detected by standard techniques. Given thehigh affinity of GRP94 for NECA, however, potential interactions of NECAwith the nucleotide binding domain could be addressed by competitivedisplacement assays. To determine the nucleotide binding specificity ofGRP94, the ability of ATP, ADP or AMP to compete with NECA binding toGRP94 was examined. In these experiments, GRP94 was incubated with 20 nM[³H]-NECA in the presence of increasing concentrations of ATP, ADP orAMP and the relative [³H]-NECA binding determined by vacuum filtration.In the presence of nominal (80 μM) Mg²⁺, it was observed that ATP, ADPand AMP effectively competed with [³H]-NECA for binding to GRP94.

Three points are evident from these experiments. One, because NECAbinding to GRP94 can be effectively inhibited by geldanamycin,radicicol, and adenosine nucleotides, it can be concluded that NECAbinds to the analogous N-terminal adenosine nucleotide binding domain ofGRP94 (FIG. 10A). Two, the relative affinities of GRP94 for ATP, ADP andAMP are quite low (FIG. 10B). Thus, a 50% inhibition of [³H]-NECAbinding required approximately a 1000-fold molar excess of ATP. Three,the relatively high binding affinity of GRP94 for NECA, when viewed withrespect to the established molecular interactions of the adenine andribose moieties of adenosine in the adenosine nucleotide binding pocketof Hsp90, suggest that a principal selection for ligands is made on thebasis of the adenosine moiety. For this reason, the interaction of otheradenosine-bearing ligands with the N-terminal nucleotide binding pocketwas examined (FIG. 10C). These data indicated that cAMP and freeadenosine also bound to the N-terminal adenosine nucleotide bindingpocket of GRP94, with the relative displacement activity approximatingthat observed for ADP.

Because the data indicated that GRP94 bound adenosine, adenosinederivatives, and adenosine nucleotides with an unusually broadspecificity, additional studies were performed to confirm the nucleosidespecificity of these binding phenomena. In the experiment depicted inFIG. 11, the [³H]-NECA competitive displacement assay was used toaddress the nucleoside base specificity directly. Though GRP94 couldbind both ATP and deoxyATP, little to no binding of GTP, CTP or UTP wasobserved. The nucleotide binding pocket of GRP94 thus appears to bestrict in its selection of adenosine-bearing ligands.

In comparing the relative affinities of GRP94 for ATP and ADP, asdisplayed in NECA competition assays, clear differences between theATP/ADP binding properties of GRP94 and those previously reported forHsp90 were noted. Regarding GRP94, ATP was found to compete NECA bindingwith an eight-fold higher efficacy than ADP. In contrast, the N-terminaldomain of Hsp90 binds ADP with a four-fold higher affinity than thatobserved for ATP (Prodromou et al. (1997) Cell 90:65-75). It washypothesized that this difference was due to a lack of Mg²⁺ ions in theassay buffer, as Mg²⁺ has been demonstrated to be essential for ATP/ADPbinding to recombinant forms of the Hsp90 N-terminal nucleotide bindingdomain by Prodromou et al. (1997) Cell 90:65-75 and Obermann et al.(1998) J Cell Biol 143:901-910.

This hypothesis was examined in experiments where the relative affinityof GRP94 for NECA, adenosine, ATP, ADP and AMP were compared in thepresence and absence of excess Mg²+(FIG. 12). In these experiments, itwas observed that although excess Mg²⁺ was without effect on the bindingof NECA or adenosine to GRP94, Mg²⁺ markedly stimulated the binding ofATP, ADP and AMP. These data are consistent with recent crystalstructure data identifying Mg²⁺ interactions with the α and β phosphatesas being requisite for ATP/ADP binding to the N-terminal domain ofHsp90. See Prodromou et al. (1997) Cell 90:65-75. However, unlike theN-terminal domain of Hsp90, MgATP and MgADP bind to GRP94 with nearlyidentical relative affinities. It should also be noted that the presenceof excess Mg²⁺ was without effect on the relative binding affinities ofcAMP and geldanamycin for GRP94.

Example 12 Nucleotide Requirement for Autophosphorylation and ATPHydrolysis

To test whether binding to the nucleotide binding pocket is directlyresponsible for the observed GRP94 autophosphorylation activity, NECAand radicicol were utilized as inhibitors of ATP binding to GRP94. Dataregarding autophosphorylation activities are shown in FIG. 13A. In thisexperiment, the autophosphorylation activity of GRP94 was assayed in thepresence of NECA, radicicol, heparin and GTP. Heparin and GTP wereincluded on the basis of previous studies indicating a casein kinaseII-like contaminant in purified preparations of GRP94 (Wearsch &Nicchitta (1997) J Biol Chem 272:5152-5156; Riera et al. (1999) Mol CellBiochem 191:97-104; and Ramakrishnan et al. (1997) J Cell Physiol170:115-29). By similar logic, the relative effects of these compoundson GRP94 kinase activity were compared in parallel with purified caseinkinase II, with casein kinase II activity measured with purified casein.

As is evident from the data presented in FIG. 13A, neither NECA norradicicol, both of which bind to the N-terminal nucleotide bindingdomain of GRP94, significantly inhibit GRP94 derived or casein kinase IIactivity below the solvent background. Because of the relatively highhydrophobicity of NECA and radicicol, incubations containing thesecompounds contained significant concentrations of the ligand solvent,dimethylsulfoxide, which itself significantly reduced both theGRP94-derived and casein kinase II activities. Heparin and GTP markedlyattenuated GRP94-derived and casein kinase II activity. In summary,blocking nucleotide access to the N-terminal adenosine nucleotide GRP94binding pocket does not significantly inhibit GRP94 autophosphorylationactivity.

The findings that cycles of ATP binding and hydrolysis function in theregulation of Hsp90 activity, and that GRP94 exhibits an ATPase activitysuggest that GRP94 and Hsp90 are indeed regulated by a similarmechanism. To further evaluate this suggestion, the ATPase activity ofGRP94 was assayed as a function of ATP concentration (FIG. 13B). Twopoints are immediately evident from the observed data. First, the ATPaseactivity does not display saturation; no evidence for a Vmax could beobtained and so traditional criteria for enzymatic function (i.e.,Km/Kcat/Vmax) could not be applied. Secondly, the absolute magnitude ofthe ATPase activity exceeded the spontaneous rate of ATP hydrolysis byonly a small factor. The observed ATPase activity was sensitive toinhibition by NECA, and thus is likely generated upon binding of ATP tothe N-terminal nucleotide binding domain.

Example 13 Conformational Consequences of Adenosine Nucleotide Bindingto GRP94

Having been unable to identify a functional correlate of ATP binding toGRP94, the effects of ATP, ADP, NECA and geldanamycin on GRP94conformation were assessed. In these studies, the tryptophan emissionspectra of GRP94, complexed with the indicated ligands, was examined asa measure of tertiary conformational state in accordance with techniquesdescribed by Guilbault (1967) Fluoresence: Theory, Instrumentation, andPractice, Marcel Dekker, Inc., New York, N.Y. As shown in FIG. 14, highconcentrations of ATP or ADP elicited near identical changes in theGRP94 tryptophan fluorescence spectra. Significantly, in the presence ofATP or ADP, the tryptophan fluorescence was decreased, as was observedin the presence of geldanamycin. These data indicate that ATP and ADPelicit a conformational change similar to that occurring in the presenceof the inhibitory ligand geldanamycin and that the conformation of GRP94in the ATP and ADP-bound state, as assessed by tryptophan fluorescence,are essentially identical. In contrast, the addition of NECA increasedthe tryptophan fluorescence, indicating that ligands can elicitdifferent conformational states in GRP94. As demonstrated in Examples1-8 above, such changes in GRP94 conformation can have dramatic effectson GRP94 chaperone function.

SUMMARY OF EXAMPLES 9-13

Examples 9-13 disclose that Hsp90 paralogs GRP94 and HSP90 displaydistinct structural and functional interactions with adenosinenucleotides. Unlike HSP90, GRP94 displays specific, high affinitybinding interactions with substituted adenosine derivatives such asN-ethylcarboxamidoadenosine (NECA). In analyzing such interactions, theoccupancy states of the N-terminal ATP/ADP binding domains of GRP94 arecommunicated between the two identical subunits. This conclusion isdrawn from the observation that at saturation NECA is bound to GRP94 ata stoichiometry of 1 mol NECA:mol GRP94 dimer. In contrast to NECA, theGRP94 inhibitory ligand, radicicol, binds at a stoichiometry of 2mol:mol GRP94. Thus, although the relevant structural components of theadenosine nucleotide binding pocket are conserved between GRP94 andHsp90, the ligand specificities of the two binding sites differ. Thus,while it is not applicants' desire to be bound by a particularlymechanistic theory, it is envisioned that the specificity of ligandbinding to the N-terminal adenosine nucleotide binding pocket isinfluenced by the domains C and N-terminal to the binding pocket, wheresignificant sequence divergence between HSP90 and GRP94 can beidentified.

The data obtained from both traditional ligand binding studies (FIG. 9)and isothermal titration calorimetry demonstrate that GRP94 binds NECAat a stoichiometry of 1 mol NECA: mol GRP94 dimer. In addition,competition studies indicate that NECA binding to GRP94 can be whollycompeted by geldanamycin, radicicol, ATP, and ADP (FIGS. 10A-10C),indicating that NECA is binding to the conserved, N-terminal adenosinenucleotide binding domain. Because GRP94 contains two such sites permolecule (Wearsch & Nicchitta (1996b) Biochemistry 35:16760-16769), itthen follows that GRP94 subunits communicate with one another to confersingle site occupancy.

The identification of ATP and ADP as the native ligands for the Hsp90proteins is based on crystallographic studies identifying an N-terminal,highly conserved nucleotide binding pocket (Prodromou et al. (1997) Cell90:65-75), complementary in vivo studies demonstrating that the aminoacids that participate in ATP/ADP binding are essential for Hsp90function in vivo and lastly (Obermann et al. (1998) J Cell Biol143:901-910; Panaretou et al. (1998) EMBO J. 17:4829-4836), that theHsp90 proteins display ATPase activity (Grenert et al. (1999) J BiolChem 274:17525-17533; Nadeau et al. (1993) J. Biol Chem 268:1479-1487;Obermann et al. (1998) J Cell Biol 143:901-910). That HSP90 and GRP94differ in NECA binding activity, despite the high homologies in theN-terminal nucleotide binding pockets of the two protein, suggests thatdifferences might also exist in the ability of the two proteins tocatalyze ATP hydrolysis. In fact, when the GRP94 ATPase activity wasinvestigated at ATP concentrations appropriate for such a low affinityinteraction it was observed that the GRP94 ATPase activity barelyexceeded the rate of spontaneous hydrolysis and, more importantly, didnot saturate at increasing ATP concentrations.

Further studies of the binding properties of the conserved domainindicated that it displays poor selectivity between adenosinenucleotides, and will bind ATP, dATP, ADP, AMP, cAMP and free adenosine.On the basis of these and other data, GRP94 conformation is regulated inan allosteric manner by an adenosine-bearing ligand other than ATP/ADP,based on ligand-mediated conformational regulation.

GRP94-dependent ATP hydrolysis, as displayed by the purified protein inthe absence of any, as yet unidentified co-factors, is non-enzymatic,and therefore unlikely to contribute to the regulation of GRP94function. Further confounding the assignment of ATP and ADP as thephysiological ligands for GRP94 are the following observations. First,neither ATP nor ADP has been demonstrated to regulate GRP94 activity, asdescribed by Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156.Secondly, that by virtue of its insensitivity to NECA and radicicol, theGRP94 autophosphorylation activity does not reflect adenosine nucleotidebinding to the N-terminal nucleotide binding domain (FIG. 13). Thirdly,and perhaps most importantly, ATP, ADP, and the inhibitor geldanamycinelicit similar conformational changes in GRP94. Interestingly, in thepresence of NECA, a different conformational change from that occurringin the presence of ATP, ADP, or geldanamycin was observed (FIG. 14).These data are consistent with ATP and ADP binding to GRP94 andstabilizing the protein in an inactive conformation, as is observed inthe presence of geldanamycin.

In evaluating these data, the inability to identify an enzymatic basisfor the ATPase activity and the conformation data suggesting thatATP/ADP would serve as inhibitory agent, either unidentified accessoryproteins interact with GRP94 to substantively alter the kinetic andthermodynamic basis for its interaction with ATP/ADP or anadenosine-based ligand, other than ATP/ADP, serves as the physiologicalligand. The ligand is produced during times of cell stress, such asanoxia, nutrient deprivation or heat shock, to activate GRP94 function.The ligand elicits a conformational change in GRP94 that substantivelyalters its interaction with substrate (poly)peptides.

Example 14 Preparation of GRP94 Ligand Binding Domain Polypeptide

Canine GRP94 69-337 (amino acids 69-337 of GENBANK® Accession No.AAA17708; residues 48-316 of SEQ ID NO: 6) was overexpressed as a GSTfusion in E. coli and purified to homogeneity by affinity andion-exchange chromatography. The protein was exchanged into 10 mMTris-HCl, pH 7.6, 1 mM DTT, 100 mM NaCl and concentrated to 30 mg/mL. Athree fold molar excess of N-ethylcarboxamidoadenosine (NECA) was addedto the protein prior to crystallization.

Example 15 Crystallization of GRP94 Ligand Binding Domain Polypeptide

Crystals were obtained by the hanging drop vapor diffusion method. Twomicroliters of GRP94 domain complexed with a three-fold molar excess ofNECA were mixed with two microliters of a reservoir solution, whichtypically comprised 100 mM Tris-HCl, pH 7.6, 150-250 mM MgCl₂, and33-38% Polyethylene glycol 400 (PEG 400). Drops were incubated at 18° C.Crystals typically formed in 2-3 days. Similar crystals could beobtained substituting PEG 1500 or PEG 4000 for the PEG 400. Twocrystalline forms, designated herein as Crystal Form 1 and Crystal Form2, were obtained.

Example 16

Crystal Data and Data Collection Statistics Crystal Form Form 1 Form 2Space group C2 C222₍₁₎ Unit cell a = 99.899 Å a = 89.200 Å b = 89.614 Åb = 99.180 Å c = 63.066 Å c = 63.071 Å β = 90.132 β = 90.0 asymmetricunit 2 GRP94 + 1 GRP94 + NECA complexes NECA complex Resolution 1.90 Å1.75 Å (last shell 2.0-1.9 Å) (1.81-1.75 Å) Completeness 97.6% (93.4%)99.7% (97.9%) Rmerge 0.117 (0.293) 0.043 (0.293) I/s(I) 17.1 (3.11) 46.9(4.45)

Example 17 Structure Solution

Crystal Forms 1 and 2 were solved by the molecular replacement method.The search model was yeast Hsp90+ADP (PDB code 1AMW). Side chains in thesearch model that were not identical to the equivalent position in GRP94were truncated to alanines.

Coordinates for Form 1 are listed in Table 1. The coordinates of theNECA and GRP94 binding pocket are listed in Table 2. Coordinates forForm 2 are listed in Table 3.

REFERENCES

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

Anderson & Matovcik (1977) Science 197:1371-1374.

Arnold et al. (1995) J Exp Med 182:885-889.

Bacalloa et al. (1994) J Cell Sci 107:3301-3313.

Basu & Srivastava (1999) J Exp Med 189:797-802.

Blachere et al. (1993) J Immunotherapy 14:352-356.

Blachere et al. (1997) J Exp Med 186:1315-1322.

Bodanszky et al. (1976) Peptide Synthesis, 2nd Ed. John Wiley & Sons.

Brawer et al. (1992) J Urol 147:841-845.

Buchner et al. (1998) Methods Enzymol 290:323-338.

Buchner (1999) Trends Biochem Sci 24:136-141.

Bumal (1988) Hybridoma 7(4):407-415.

Caplan (1999) Trends Cell Biol 9:262-268.

Catalona et al. (1993) JAMA 270:948-958.

Chadli et al. (1999) J Biol Chem 274:4133-4139.

Chang et al. (1997) Mol Cell Biol 17:318-25.

Chavany et al. (1996) J Biol Chem 271:4974-4977.

Chen et al. (1996) Mol Endocrinol 10:682-693.

Chen et al. (1996) J Cereb Blood Flow Metab 16:566-577.

Chien et al. (1991) Proc Natl Acad Sci USA 88:9578-9582.

Choi et al. (1987) J Neurosci 7:357.

Csermely & Kahn (1991) J Biol Chem 266:4943-4950.

Csermely et al. (1995) J Biol Chem 270:6381-6388.

Csermely et al. (1993) J Biol Chem 268:1901-1907.

Csermely et al. (1998) Pharmacol Ther 79:129-168.

Davis & Maher (1994) Brain Res 652(1):169-173.

Demotz et al. (1989) Nature 343:682-684.

Dittmar et al. (1998) J Biol Chem 273:7358-7366.

Doherty et al. (1995) Neuron 14:57-66.

Duina et al. (1996) Science 274:1713-1715.

Elliott et al. (1990) Nature 348:191-197.

Falk et al. (1991) Nature 351:290-296.

Falk et al. (1990) Nature 348:248-251.

Fan et al. (1999) J Mol Med 77:577-596.

Ferreira et al. (1994) J Cell Biochem 56:518-26.

Fields et al. (1990) Int J Peptide Protein Res 35:161-214.

Flynn et al. (1989) Science 245:385-390.

Freireich et al. (1966) Cancer Chemotherap Rep 50:219-244.

Gerweck et al. (1979) Cancer Res 39:966-972.

Ginsberg & Busto (1989) Stroke 20:1627.

Glasebrook et al. (1980) J Exp Med 151:876.

Gradin et al. (1996) Mol Cell Biol 16:5221-5231.

Grenert et al. (1999) J Biol Chem 274:17525-17533.

Grollman et al. (1993) J Biol Chem 268:3604-3609.

Hansen et al. (1989) Electrophoresis 10:645-652.

Hebert et al. (1996) EMBO J. 15:2961-2968.

Hebert et al. (1997) J Cell Biol 139:613-623.

Heike et al. (1996) J Leukoc Biol 60:153-8.

Heike et al. (1994) J Immunotherapy 15:165-174.

Henttu & Vihko (1989) Biochem Biophys Res Comm 160(2):903-910.

Horch et al. (1999) Neuron 23:353-364.

Hutchison et al. (1990) Biochemistry 29:5138-5144.

Hutchison & Fox (1989) J Biol Chem 264:19898-19903.

Huttemann et al. (1984) Naunyn Schmiedebergs Arch Pharmacol 325:226-233.

Inaba (1992) J Exp Med 176:1693-1702.

Ishii et al. (1999) J Immunol 162:1303-1309.

Israeli et al. (1993) Cancer Res 53:227-230.

Jakob et al. (1995) J Biol Chem 270:7288-7294.

Jakob et al. (1996) J Biol Chem 271:10035-10041.

Johnson et al. (1996) J Steroid Biochem Mol Biol 56:31-37.

Karpiak et al. (1989) Ann Rev Pharmacol Toxicol 29:403.

Kassenbrock & Kelly (1989) EMBO J. 8:1461-1467.

Kosano et al. (1998) J Biol Chem 273:32973-32979.

Kuznetsov et al. (1994) J Biol Chem 269:22990-22995.

Kuznetsov (1996) Proc Natl Acad Sci USA 93:8584-8589.

Lawson et al. (1998) J Cell Physiol 174:170-178.

Li & Srivastava (1993) EMBO J. 12:3143-3151.

Li et al. (1993) EMBO J. 12:3143-3151.

Mandel et al. (1994) J Cell Sci 107:3315-224.

Masliah et al. (1992) Exp Neurol 136:107-122.

Massa et al. (1996) “The Stress Gene Response in Brain” inCerebrovascular and Brain Metabolism Reviews, pp. 95-158,Lippincott-Raven Publishers, Philadelphia, Pa.

McAllister et al. (1997) Neuron 18:767-778.

McAuley (1995) Cerebrovasc Brain Metab Review 7:153-180.

McOmie (1973) Protective Groups in Organic Chemistry, Plenum Press, NewYork, N.Y.

Meienhofer (1983) Hormonal Proteins and Peptides Vol. 2, pp. 46,Academic Press, New York, N.Y.

Melnick et al. (1992) J Biol Chem 267:21303-21306.

Melnick et al. (1994) Nature 370:373-375.

Merrifield (1969) Adv Enzymol 32:221-296.

Microcal Software (1998) MicroCal ORIGINTM, MicroCal Inc., Northhampton,Mass.

Mitchell et al. (1998) Eur J Immunol 28:1923-1933.

Mizoe et al. (1997) J Surg Res 73(2):160-165.

Myers & Jakoby (1975) J Biol Chem 250:3785-3789.

Nadeau et al. (1993) J Biol Chem 268:1479-1487.

Nair et al. (1999) J Immunol 162:6426-6432.

Natali et al. (1987) Cancer 59:55-63.

Navarro et al. (1991) Virology 184:253-264.

Nemoto et al. (1996) J Biochem 120:249-256.

Nicchitta (1998) Curr Opin Immunol 10:103-109.

Nieland et al. (1996) Proc Natl Acad Sci USA 93:6135-6139.

Norrby (1985) “Summary” in Vaccines 85, Lerner et al. (eds.), pp.388-389, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

Obermann et al. (1998) J Cell Biol 143:901-910.

Ortmann et al. (1997) Science 277:1306-1309.

Palladino et al. (1987) Cancer Res 47:5074-5079.

Palleros et al. (1991) Proc Natl Acad Sci USA 88:5719-5723.

Panaretou et al. (1998) EMBO J. 17:4829-4836.

Perez & Walker (1990) J Immunol 142:3662-3667.

Pratt (1998) Proc Soc Exp Biol Med 217:420-434.

Pratt et al. (1996) Exs 77:79-95.

Prodromou et al. (1999) EMBO J. 18:754-762.

Prodromou et al. (1997) Cell 90:65-75.

Ramachandran & Gottlieb (1961) Biochim Biophys Acta 53:396-402.

Ramakrishnan et al. (1997) J Cell Physiol 170:115-29.

Riera et al. (1999) Mol Cell Biochem 191:97-104.

Robbins & Angell (1976) Basic Pathology, 2nd Ed., pp. 68-79, W. B.Saunders Co., Philadelphia, Pa.

Roe et al. (1999) J Med Chem 42:260-266.

Rose et al. (1987) Biochemistry 26:6583-6587.

Rosen & Weber (1969) Biochemistry 8:3915-3920.

Rotzsche et al. (1990) Nature 348:252-254.

Rotzsche et al. (1990) Science 249:283-287.

Sadasivan et al. (1996) Cell 5:103-114.

Sastry & Linderoth (1999) J Biol Chem 274:12023-12035.

Sato et al. (1995) Clin Immunol Pathol 74:35-43.

Schagger et al. (1994) Anal Biochem 217:220-230.

Schaiff et al. (1992) J Exp Med 176:657-666.

Scheibel & Buckner (1998) Biochem Pharm 56:675-82.

Scheibel et al. (1998) Proc Natl Acad Sci USA 95:1495-1499.

Scheibel et al. (1997) J Biol Chem 272:18608-18613.

Schild et al. (1999) Curr Opin Immunol 11:109-113.

Schnell et al. (1990) Nature 343:269-272.

Schroder et al. (1965) The Peptides, Vol. 1, Academic Press, New York,N.Y.

Schroder et al. (1965) JAMA 193:443.

Sciandra et al. (1984) Proc Natl Acad Sci USA 81:4843-4847.

Seale et al. (1998) Methods Enzymol 290:318-323.

Seip & Evans (1980) J Clin Microbiol 11:226-233.

Sharma et al. (1998) J Biol Chem 273:15474-15478.

Sharma et al. (1998) Oncogene 16:2639-2645.

Shi et al. (1994) Biochemistry 33:7536-7546.

Shirkey (1965) JAMA 193:443.

Smith et al. (1993) J Biol Chem 268:18365-18371.

Sriram et al. (1997) Structure 5:403-414.

Srivastava et al. (1986) Proc Natl Acad Sci USA 83:3407-3411.

Srivastava et al. (1998) Immunity 8:657-665.

Srivastava et al. (1994) Immunogenetics 39:93-98.

Stebbins et al. (1997) Cell 89:239-250.

Steinman (1991) Annu Rev Immunol 9:271-294.

Steward et al. (1969) Solid Phase Peptide Synthesis, W. H. Freeman Co.,San Francisco, Calif.

Su et al. (1998) J Mol Cell Cardiol 30(3):587-598.

Sullivan et al. (1997) J Biol Chem 272:8007-8012.

Supino-Rosin et al. (2000) J Biol Chem 275(29):21850-21855.

Suto & Srivastava (1995) Science 269:1585-1588.

Tacchini et al. (1997) Hepatology 26(1): 186-191.

Tailer et al. (1990) Nuc Acids Res 18(16):4928.

Takashi et al. (1977) Proc Natl Acad Sci USA 74:2334-2338.

Tamura et al. (1997) Science 278:117-120.

Toft (1998) Trends Endocrinol Metab 9:238-243.

Toggas et al. (1994) Nature 367:188-193.

Udono et al. (1994) Proc Natl Acad Sci USA 91:3077-81.

U.S. Pat. No. 4,244,946

U.S. Pat. No. 4,968,671

U.S. Pat. No. 5,066,578

U.S. Pat. No. 5,250,414

U.S. Pat. No. 5,352,660

U.S. Pat. No. 5,504,090

U.S. Pat. No. 5,645,999

U.S. Pat. No. 5,571,840

U.S. Pat. No. 5,733,916

U.S. Pat. No. 5,739,278

U.S. Pat. No. 5,747,332

U.S. Pat. No. 5,750,119

U.S. Pat. No. 5,756,492

U.S. Pat. No. 5,786,152

U.S. Pat. No. 5,830,464

U.S. Pat. No. 5,834,228

U.S. Pat. No. 5,837,251

U.S. Pat. No. 5,872,011

U.S. Pat. No. 5,898,066

U.S. Pat. No. 5,932,542

U.S. Pat. No. 6,017,965

U.S. Pat. No. 6,046,381

U.S. Pat. No. 6,080,730

Van Bleek et al. (1990) Nature 348:213-216.

Vijayasardahl et al. (1990) J Exp Med 171(4):1375-1380.

Wearsch & Nicchitta (1996a) Prot Express Purif 7:114-121.

Wearsch & Nicchitta (1996b) Biochemistry 35:16760-16769.

Wearsch & Nicchitta (1997) J Biol Chem 272:5152-5156.

Wearsch et al. (1998) Biochemistry 37:5709-5719.

Weber (1991) Adv Protein Chem 41:1-36.

Weber & Farris (1979) Biochemistry 18:3075-3078.

WO 95/24923

WO 97/10000

WO 97/10002

WO 98/34641

WO 99/26966

WO 99/61585

Xiao et al. (1999) J Neurochem 72:95-101.

Yagita et al. (1999) J Neurochem 72:1544-1551.

Yamamoto et al. (1986) Brain Res 384:1-10.

Yamamoto et al. (1990) Acta Neuropathol 80:487-492.

Yu et al. (1991) Cancer Res 51(2):468-475.

Zimmer et al. (1993) Peptides, pp. 393-394, ESCOM Science Publishers, B.V.

TABLE 1 ATOMIC STRUCTURE COORDINATE DATA OBTAINED FROM X-RAY DIFFRACTIONFROM FORM 1 OF THE LIGAND BINDING DOMAIN OF GRP94 (RESIDUES 48-316 OFSEQ ID NO: 6) IN COMPLEX WITH NECA ATOM 1 CB LYS A 72 16.433 13.81340.193 1.00 72.11 A ATOM 2 CG LYS A 72 15.697 13.996 41.523 1.00 73.17 AATOM 3 CD LYS A 72 14.969 12.738 41.966 1.00 74.15 A ATOM 4 CE LYS A 7214.239 12.957 43.285 1.00 74.93 A ATOM 5 NZ LYS A 72 15.159 13.35544.391 1.00 76.86 A ATOM 6 C LYS A 72 15.008 12.263 38.820 1.00 70.47 AATOM 7 O LYS A 72 15.763 11.359 38.463 1.00 70.77 A ATOM 8 N LYS A 7214.412 14.682 39.050 1.00 71.64 A ATOM 9 CA LYS A 72 15.525 13.69438.960 1.00 71.44 A ATOM 10 N SER A 73 13.719 12.076 39.102 1.00 68.65 AATOM 11 CA SER A 73 13.064 10.771 39.014 1.00 67.10 A ATOM 12 CB SER A73 12.943 10.149 40.409 1.00 67.99 A ATOM 13 OG SER A 73 12.307 8.88440.354 1.00 70.18 A ATOM 14 C SER A 73 11.672 10.963 38.411 1.00 64.75 AATOM 15 O SER A 73 10.948 11.878 38.810 1.00 66.63 A ATOM 16 N GLU A 7411.294 10.112 37.458 1.00 59.74 A ATOM 17 CA GLU A 74 9.988 10.23836.813 1.00 56.73 A ATOM 18 CB GLU A 74 10.126 11.049 35.515 1.00 59.39A ATOM 19 CG GLU A 74 8.809 11.498 34.890 1.00 63.49 A ATOM 20 CD GLU A74 8.144 12.641 35.648 1.00 67.43 A ATOM 21 OE1 GLU A 74 7.003 13.01335.285 1.00 67.96 A ATOM 22 OE2 GLU A 74 8.762 13.172 36.599 1.00 69.08A ATOM 23 C GLU A 74 9.352 8.876 36.513 1.00 53.34 A ATOM 24 O GLU A 7410.030 7.938 36.087 1.00 48.79 A ATOM 25 N ALA A 75 8.046 8.781 36.7391.00 50.06 A ATOM 26 CA ALA A 75 7.307 7.542 36.510 1.00 48.67 A ATOM 27CB ALA A 75 6.332 7.298 37.666 1.00 48.93 A ATOM 28 C ALA A 75 6.5517.586 35.188 1.00 47.67 A ATOM 29 O ALA A 75 6.008 8.630 34.802 1.0046.57 A ATOM 30 N PHE A 76 6.518 6.454 34.489 1.00 43.49 A ATOM 31 CAPHE A 76 5.817 6.381 33.214 1.00 42.64 A ATOM 32 CB PHE A 76 6.795 6.39832.035 1.00 43.68 A ATOM 33 CG PHE A 76 7.708 7.593 31.986 1.00 43.05 AATOM 34 CD1 PHE A 76 8.843 7.656 32.793 1.00 44.22 A ATOM 35 CD2 PHE A76 7.456 8.630 31.088 1.00 44.40 A ATOM 36 CE1 PHE A 76 9.726 8.74032.704 1.00 46.28 A ATOM 37 CE2 PHE A 76 8.331 9.719 30.989 1.00 44.49 AATOM 38 CZ PHE A 76 9.468 9.772 31.798 1.00 43.57 A ATOM 39 C PHE A 764.994 5.104 33.063 1.00 43.02 A ATOM 40 O PHE A 76 5.291 4.076 33.6781.00 41.13 A ATOM 41 N ALA A 77 3.971 5.181 32.222 1.00 42.30 A ATOM 42CA ALA A 77 3.153 4.021 31.909 1.00 40.65 A ATOM 43 CB ALA A 77 1.6824.393 31.872 1.00 41.85 A ATOM 44 C ALA A 77 3.633 3.659 30.513 1.0040.75 A ATOM 45 O ALA A 77 4.072 4.544 29.761 1.00 36.27 A ATOM 46 N PHEA 78 3.593 2.374 30.164 1.00 38.04 A ATOM 47 CA PHE A 78 4.000 1.97728.819 1.00 37.01 A ATOM 48 CB PHE A 78 4.184 0.456 28.702 1.00 32.60 AATOM 49 CG PHE A 78 5.427 −0.073 29.350 1.00 31.73 A ATOM 50 CD1 PHE A78 5.405 −0.544 30.668 1.00 33.46 A ATOM 51 CD2 PHE A 78 6.606 −0.17128.624 1.00 30.57 A ATOM 52 CE1 PHE A 78 6.548 −1.115 31.246 1.00 28.34A ATOM 53 CE2 PHE A 78 7.750 −0.736 29.188 1.00 28.60 A ATOM 54 CZ PHE A78 7.719 −1.211 30.503 1.00 29.55 A ATOM 55 C PHE A 78 2.877 2.37627.868 1.00 36.00 A ATOM 56 O PHE A 78 1.728 2.522 28.281 1.00 36.79 AATOM 57 N GLN A 79 3.209 2.549 26.595 1.00 36.10 A ATOM 58 CA GLN A 792.204 2.876 25.597 1.00 38.02 A ATOM 59 CB GLN A 79 2.871 3.230 24.2741.00 38.53 A ATOM 60 CG GLN A 79 1.920 3.686 23.185 1.00 42.76 A ATOM 61CD GLN A 79 2.622 3.875 21.855 1.00 45.14 A ATOM 62 OE1 GLN A 79 3.8184.172 21.813 1.00 45.16 A ATOM 63 NE2 GLN A 79 1.879 3.718 20.760 1.0044.85 A ATOM 64 C GLN A 79 1.384 1.590 25.444 1.00 38.00 A ATOM 65 O GLNA 79 1.910 0.488 25.642 1.00 36.49 A ATOM 66 N ALA A 80 0.109 1.73225.110 1.00 37.16 A ATOM 67 CA ALA A 80 −0.774 0.578 24.953 1.00 38.95 AATOM 68 CB ALA A 80 −2.124 1.029 24.389 1.00 38.25 A ATOM 69 C ALA A 80−0.179 −0.512 24.058 1.00 39.42 A ATOM 70 O ALA A 80 −0.120 −1.68424.444 1.00 41.54 A ATOM 71 N GLU A 81 0.256 −0.126 22.862 1.00 39.30 AATOM 72 CA GLU A 81 0.820 −1.077 21.914 1.00 39.92 A ATOM 73 CB GLU A 811.234 −0.370 20.622 1.00 43.06 A ATOM 74 CG GLU A 81 0.054 0.097 19.7761.00 48.76 A ATOM 75 CD GLU A 81 −0.613 1.358 20.313 1.00 51.87 A ATOM76 OE1 GLU A 81 −1.641 1.779 19.734 1.00 53.98 A ATOM 77 OE2 GLU A 81−0.108 1.930 21.307 1.00 52.54 A ATOM 78 C GLU A 81 1.994 −1.854 22.4801.00 38.84 A ATOM 79 O GLU A 81 2.175 −3.034 22.159 1.00 38.13 A ATOM 80N VAL A 82 2.791 −1.203 23.319 1.00 35.57 A ATOM 81 CA VAL A 82 3.934−1.869 23.925 1.00 34.33 A ATOM 82 CB VAL A 82 4.868 −0.851 24.623 1.0033.22 A ATOM 83 CG1 VAL A 82 5.948 −1.579 25.385 1.00 32.87 A ATOM 84CG2 VAL A 82 5.513 0.069 23.575 1.00 32.40 A ATOM 85 C VAL A 82 3.453−2.936 24.921 1.00 34.76 A ATOM 86 O VAL A 82 4.066 −4.1.003 25.047 1.0033.56 A ATOM 87 N ASN A 83 2.362 −2.658 25.628 1.00 33.27 A ATOM 88 CAASN A 83 1.832 −3.655 26.553 1.00 34.85 A ATOM 89 CB ASN A 83 0.635−3.119 27.344 1.00 34.31 A ATOM 90 CG ASN A 83 1.039 −2.186 28.475 1.0035.50 A ATOM 91 OD1 ASN A 83 2.087 −2.353 29.100 1.00 33.31 A ATOM 92ND2 ASN A 83 0.183 −1.210 28.762 1.00 32.10 A ATOM 93 C ASN A 83 1.390−4.874 25.731 1.00 36.41 A ATOM 94 O ASN A 83 1.693 −6.016 26.086 1.0036.00 A ATOM 95 N ARG A 84 0.690 −4.623 24.629 1.00 36.92 A ATOM 96 CAARG A 84 0.223 −5.693 23.741 1.00 38.05 A ATOM 97 CB ARG A 84 −0.626−5.118 22.604 1.00 40.19 A ATOM 98 CG ARG A 84 −1.773 −4.231 23.077 1.0044.63 A ATOM 99 CD ARG A 84 −2.814 −3.974 21.989 1.00 47.27 A ATOM 100NE ARG A 84 −3.925 −3.180 22.520 1.00 49.29 A ATOM 101 CZ ARG A 84−4.066 −1.868 22.348 1.00 50.38 A ATOM 102 NH1 ARG A 84 −3.175 −1.18421.642 1.00 50.19 A ATOM 103 NH2 ARG A 84 −5.088 −1.236 22.910 1.0050.47 A ATOM 104 C ARG A 84 1.414 −6.449 23.145 1.00 39.01 A ATOM 105 OARG A 84 1.393 −7.684 23.042 1.00 38.93 A ATOM 106 N MET A 85 2.451−5.703 22.762 1.00 36.19 A ATOM 107 CA MET A 85 3.652 −6.287 22.182 1.0036.75 A ATOM 108 CB MET A 85 4.609 −5.184 21.693 1.00 38.82 A ATOM 109CG MET A 85 5.974 −5.703 21.210 1.00 41.23 A ATOM 110 SD MET A 85 6.976−4.519 20.233 1.00 43.17 A ATOM 111 CE MET A 85 7.035 −3.128 21.369 1.0045.81 A ATOM 112 C MET A 85 4.374 −7.186 23.177 1.00 36.50 A ATOM 113 OMET A 85 4.917 −8.229 22.797 1.00 37.23 A ATOM 114 N MET A 86 4.393−6.778 24.443 1.00 34.68 A ATOM 115 CA MET A 86 5.052 −7.557 25.477 1.0036.95 A ATOM 116 CB MET A 86 5.041 −6.811 26.822 1.00 36.65 A ATOM 117CG MET A 86 6.019 −5.622 26.896 1.00 37.30 A ATOM 118 SD MET A 86 6.348−5.091 28.589 1.00 39.30 A ATOM 119 CE MET A 86 5.046 −3.884 28.822 1.0041.95 A ATOM 120 C MET A 86 4.352 −8.905 25.617 1.00 37.51 A ATOM 121 OMET A 86 5.007 −9.944 25.651 1.00 38.48 A ATOM 122 N ALA A 87 3.025−8.876 25.694 1.00 37.56 A ATOM 123 CA ALA A 87 2.227 −10.097 25.8331.00 38.50 A ATOM 124 CB ALA A 87 0.752 −9.736 25.976 1.00 36.26 A ATOM125 C ALA A 87 2.429 −11.057 24.651 1.00 39.16 A ATOM 126 O ALA A 872.533 −12.278 24.839 1.00 40.78 A ATOM 127 N LEU A 88 2.485 −10.50723.442 1.00 39.19 A ATOM 128 CA LEU A 88 2.682 −11.310 22.232 1.00 41.17A ATOM 129 CB LEU A 88 2.613 −10.429 20.984 1.00 43.80 A ATOM 130 CG LEUA 88 1.256 −9.839 20.609 1.00 46.16 A ATOM 131 CD1 LEU A 88 1.421 −8.94519.379 1.00 48.50 A ATOM 132 CD2 LEU A 88 0.255 −10.968 20.337 1.0048.36 A ATOM 133 C LEU A 88 4.030 −12.015 22.254 1.00 41.23 A ATOM 134 OLEU A 88 4.136 −13.199 21.935 1.00 40.24 A ATOM 135 N ILE A 89 5.065−11.276 22.629 1.00 39.12 A ATOM 136 CA ILE A 89 6.410 −11.825 22.6911.00 38.88 A ATOM 137 CB ILE A 89 7.439 −10.714 22.993 1.00 37.36 A ATOM138 CG2 ILE A 89 8.788 −11.323 23.330 1.00 33.69 A ATOM 139 CG1 ILE A 897.533 −9.765 21.798 1.00 36.49 A ATOM 140 CD1 ILE A 89 8.545 −8.63021.990 1.00 35.13 A ATOM 141 C ILE A 89 6.508 −12.893 23.766 1.00 40.48A ATOM 142 O ILE A 89 6.997 −14.004 23.523 1.00 41.38 A ATOM 143 N ILE A90 6.032 −12.551 24.953 1.00 39.66 A ATOM 144 CA ILE A 90 6.071 −13.46326.078 1.00 44.50 A ATOM 145 CB ILE A 90 5.397 −12.825 27.299 1.00 45.01A ATOM 146 CG2 ILE A 90 5.148 −13.876 28.374 1.00 46.20 A ATOM 147 CG1ILE A 90 6.266 −11.674 27.814 1.00 47.20 A ATOM 148 CD1 ILE A 90 5.599−10.830 28.891 1.00 48.97 A ATOM 149 C ILE A 90 5.387 −14.789 25.7441.00 46.87 A ATOM 150 O ILE A 90 5.985 −15.857 25.896 1.00 46.46 A ATOM151 N ASN A 91 4.142 −14.709 25.283 1.00 48.34 A ATOM 152 CA ASN A 913.369 −15.900 24.931 1.00 51.26 A ATOM 153 CB ASN A 91 1.936 −15.50424.562 1.00 53.61 A ATOM 154 CG ASN A 91 1.047 −15.322 25.779 1.00 57.42A ATOM 155 OD1 ASN A 91 0.704 −16.292 26.460 1.00 60.21 A ATOM 156 ND2ASN A 91 0.668 −14.079 26.062 1.00 58.87 A ATOM 157 C ASN A 91 3.995−16.685 23.782 1.00 50.90 A ATOM 158 O ASN A 91 3.879 −17.908 23.7231.00 51.42 A ATOM 159 N SER A 92 4.664 −15.978 22.877 1.00 50.08 A ATOM160 CA SER A 92 5.306 −16.601 21.726 1.00 49.59 A ATOM 161 CB SER A 925.700 −15.528 20.710 1.00 52.19 A ATOM 162 OG SER A 92 6.570 −16.05519.721 1.00 55.91 A ATOM 163 C SER A 92 6.540 −17.425 22.091 1.00 48.23A ATOM 164 O SER A 92 6.792 −18.476 21.495 1.00 48.01 A ATOM 165 N LEU A93 7.302 −16.946 23.071 1.00 44.77 A ATOM 166 CA LEU A 93 8.523 −17.61223.513 1.00 44.30 A ATOM 167 CB LEU A 93 9.667 −16.585 23.569 1.00 44.17A ATOM 168 CG LEU A 93 9.955 −15.801 22.285 1.00 45.55 A ATOM 169 CD1LEU A 93 10.928 14.665 22.576 1.00 45.11 A ATOM 170 CD2 LEU A 93 10.521−16.746 21.221 1.00 46.15 A ATOM 171 C LEU A 93 8.383 −18.286 24.8851.00 40.97 A ATOM 172 O LEU A 93 9.378 −18.660 25.500 1.00 39.89 A ATOM173 N TYR A 94 7.155 −18.451 25.358 1.00 41.99 A ATOM 174 CA TYR A 946.929 −19.049 26.670 1.00 43.74 A ATOM 175 CB TYR A 94 5.430 −19.19926.930 1.00 46.24 A ATOM 176 CG TYR A 94 5.109 −19.509 28.371 1.00 51.80A ATOM 177 CD1 TYR A 94 5.315 −18.555 29.372 1.00 53.42 A ATOM 178 CE1TYR A 94 5.046 −18.842 30.707 1.00 55.59 A ATOM 179 CD2 TYR A 94 4.622−20.762 28.742 1.00 53.62 A ATOM 180 CE2 TYR A 94 4.347 −21.061 30.0751.00 55.21 A ATOM 181 CZ TYR A 94 4.562 −20.099 31.053 1.00 56.87 A ATOM182 OH TYR A 94 4.304 −20.393 32.378 1.00 57.87 A ATOM 183 C TYR A 947.621 −20.402 26.875 1.00 44.70 A ATOM 184 O TYR A 94 8.070 −20.71027.981 1.00 42.57 A ATOM 185 N LYS A 95 7.723 −21.193 25.808 1.00 45.14A ATOM 186 CA LYS A 95 8.345 22.518 25.886 1.00 46.47 A ATOM 187 CB LYSA 95 7.562 −23.504 25.020 1.00 48.12 A ATOM 188 CG LYS A 95 6.164−23.818 25.523 1.00 52.83 A ATOM 189 CD LYS A 95 6.210 −24.688 26.7701.00 57.04 A ATOM 190 CE LYS A 95 4.848 −25.304 27.080 1.00 59.01 A ATOM191 NZ LYS A 95 3.827 −24.288 27.471 1.00 60.76 A ATOM 192 C LYS A 959.818 −22.558 25.484 1.00 46.81 A ATOM 193 O LYS A 95 10.442 −23.62825.479 1.00 45.94 A ATOM 194 N ASN A 96 10.368 −21.396 25.147 1.00 44.69A ATOM 195 CA ASN A 96 11.766 −21.282 24.740 1.00 44.51 A ATOM 196 CBASN A 96 11.855 −21.178 23.216 1.00 47.19 A ATOM 197 CG ASN A 96 11.311−22.410 22.519 1.00 49.13 A ATOM 198 OD1 ASN A 96 11.959 −23.458 22.4921.00 51.66 A ATOM 199 ND2 ASN A 96 10.114 −22.295 21.961 1.00 49.94 AATOM 200 C ASN A 96 12.333 −20.022 25.385 1.00 44.78 A ATOM 201 O ASN A96 12.886 −19.153 24.712 1.00 41.88 A ATOM 202 N LYS A 97 12.198 −19.94926.703 1.00 43.79 A ATOM 203 CA LYS A 97 12.638 −18.792 27.471 1.0043.72 A ATOM 204 CB LYS A 97 12.351 −19.027 28.960 1.00 42.66 A ATOM 205CG LYS A 97 10.877 −19.256 29.288 1.00 43.52 A ATOM 206 CD LYS A 9710.671 −19.362 30.800 1.00 45.04 A ATOM 207 CE LYS A 97 9.211 −19.23731.205 1.00 47.13 A ATOM 208 NZ LYS A 97 8.324 −20.293 30.652 1.00 46.59A ATOM 209 C LYS A 97 14.089 −18.362 27.292 1.00 43.92 A ATOM 210 O LYSA 97 14.387 −17.166 27.357 1.00 43.02 A ATOM 211 N GLU A 98 14.990−19.313 27.060 1.00 41.72 A ATOM 212 CA GLU A 98 16.402 −18.979 26.9041.00 42.63 A ATOM 213 CB GLU A 98 17.242 −20.245 26.693 1.00 43.53 AATOM 214 CG GLU A 98 16.859 −21.079 25.483 1.00 49.13 A ATOM 215 CD GLUA 98 15.763 −22.095 25.779 1.00 51.40 A ATOM 216 OE1 GLU A 98 15.433−22.891 24.872 1.00 54.63 A ATOM 217 OE2 GLU A 98 15.234 −22.102 26.9111.00 51.95 A ATOM 218 C GLU A 98 16.712 −17.983 25.787 1.00 40.21 A ATOM219 O GLU A 98 17.764 −17.350 25.788 1.00 40.18 A ATOM 220 N ILE A 9915.803 −17.847 24.834 1.00 40.55 A ATOM 221 CA ILE A 99 16.009 −16.92423.727 1.00 41.11 A ATOM 222 CB ILE A 99 14.856 −17.053 22.703 1.0043.78 A ATOM 223 CG2 ILE A 99 14.851 −15.871 21 735 1.00 44.38 A ATOM224 CG1 ILE A 99 15.022 −18.364 21.921 1.00 45.95 A ATOM 225 CD1 ILE A99 13.993 −18.570 20.823 1.00 46.27 A ATOM 226 C ILE A 99 16.145 −15.47524.216 1.00 40.18 A ATOM 227 O ILE A 99 16.583 −14.589 23.467 1.00 37.76A ATOM 228 N PHE A 100 15.794 −15.234 25.475 1.00 38.91 A ATOM 229 CAPHE A 100 15.905 −13.881 26.009 1.00 39.16 A ATOM 230 CB PHE A 10015.387 −13.800 27.455 1.00 36.23 A ATOM 231 CG PHE A 100 16.407 −14.17928.495 1.00 37.78 A ATOM 232 CD1 PHE A 100 16.516 −15.497 28.936 1.0036.00 A ATOM 233 CD2 PHE A 100 17.264 −13.218 29.034 1.00 35.49 A ATOM234 CE1 PHE A 100 17.456 −15.851 29.894 1.00 34.24 A ATOM 235 CE2 PHE A100 18.210 −13.563 29.995 1.00 36.56 A ATOM 236 CZ PHE A 100 18.309−14.880 30.427 1.00 39.89 A ATOM 237 C PHE A 100 17.364 −13.449 25.9581.00 38.09 A ATOM 238 O PHE A 100 17.664 −12.292 25.679 1.00 37.97 AATOM 239 N LEU A 101 18.269 −14.390 26.216 1.00 37.51 A ATOM 240 CA LEUA 101 19.693 −14.087 26.219 1.00 38.15 A ATOM 241 CB LEU A 101 20.480−15.213 26.901 1.00 39.85 A ATOM 242 CG LEU A 101 21.961 −14.918 27.1711.00 41.99 A ATOM 243 CD1 LEU A 101 22.091 −13.710 28.100 1.00 42.72 AATOM 244 CD2 LEU A 101 22.624 −16.130 27.795 1.00 43.76 A ATOM 245 C LEUA 101 20.227 −13.854 24.808 1.00 37.64 A ATOM 246 O LEU A 101 21.167−13.079 24.607 1.00 36.64 A ATOM 247 N ARG A 102 19.640 −14.537 23.8331.00 35.76 A ATOM 248 CA ARG A 102 20.050 −14.369 22.442 1.00 36.99 AATOM 249 CB ARG A 102 19.245 −15.298 21.533 1.00 37.21 A ATOM 250 CG ARGA 102 19.738 −15.335 20.092 1.00 41.86 A ATOM 251 CD ARG A 102 18.773−16.104 19.196 1.00 41.14 A ATOM 252 NE ARG A 102 17.589 −15.320 18.8621.00 40.14 A ATOM 253 CZ ARG A 102 16.555 −15.785 18.167 1.00 45.54 AATOM 254 NH1 ARG A 102 16.560 −17.039 17.732 1.00 44.26 A ATOM 255 NH2ARG A 102 15.515 −14.998 17.903 1.00 43.91 A ATOM 256 C ARG A 102 19.770−12.922 22.032 1.00 36.16 A ATOM 257 O ARG A 102 20.607 −12.266 21.4141.00 34.66 A ATOM 258 N GLU A 103 18.584 −12.439 22.390 1.00 35.80 AATOM 259 CA GLU A 103 18.172 −11.084 22.033 1.00 36.77 A ATOM 260 CB GLUA 103 16.667 −10.927 22.244 1.00 38.94 A ATOM 261 CG GLU A 103 15.845−11.797 21.293 1.00 44.14 A ATOM 262 CD GLU A 103 16.277 −11.613 19.8431.00 48.10 A ATOM 263 OE1 GLU A 103 16.200 −10.471 19.344 1.00 47.73 AATOM 264 OE2 GLU A 103 16.706 −12.603 19.205 1.00 48.75 A ATOM 265 C GLUA 103 18.933 −9.987 22.764 1.00 37.18 A ATOM 266 O GLU A 103 19.260−8.956 22.167 1.00 38.35 A ATOM 267 N LEU A 104 19.213 −10.181 24.0521.00 36.53 A ATOM 268 CA LEU A 104 19.967 −9.168 24.782 1.00 34.96 AATOM 269 CB LEU A 104 20.051 −9.514 26.266 1.00 36.16 A ATOM 270 CG LEUA 104 18.724 −9.575 27.021 1.00 34.61 A ATOM 271 CD1 LEU A 104 18.994−9.853 28.490 1.00 36.29 A ATOM 272 CD2 LEU A 104 17.971 −8.254 26.8551.00 37.01 A ATOM 273 C LEU A 104 21.369 −9.081 24.183 1.00 36.79 A ATOM274 O LEU A 104 21.956 −7.995 24.098 1.00 35.24 A ATOM 275 N ILE A 10521.916 −10.223 23.765 1.00 34.90 A ATOM 276 CA ILE A 105 23.241 −10.21723.164 1.00 35.92 A ATOM 277 CB ILE A 105 23.819 −11.649 23.074 1.0036.84 A ATOM 278 CG2 ILE A 105 24.983 −11.688 22.095 1.00 37.43 A ATOM279 CG1 ILE A 105 24.260 −12.106 24.474 1.00 39.98 A ATOM 280 CD1 ILE A105 24.758 −13.546 24.536 1.00 41.86 A ATOM 281 C ILE A 105 23.184−9.559 21.781 1.00 36.00 A ATOM 282 O ILE A 105 24.082 −8.795 21.4151.00 36.27 A ATOM 283 N SER A 106 22.118 −9.833 21.032 1.00 36.13 A ATOM284 CA SER A 106 21.944 −9.231 19.715 1.00 37.12 A ATOM 285 CB SER A 10620.715 −9.822 19.029 1.00 38.68 A ATOM 286 OG SER A 106 20.542 −9.25817.740 1.00 45.19 A ATOM 287 C SER A 106 21.785 −7.704 19.866 1.00 37.64A ATOM 288 O SER A 106 22.343 −6.921 19.085 1.00 35.27 A ATOM 289 N ASNA 107 21.038 −7.279 20.880 1.00 35.94 A ATOM 290 CA ASN A 107 20.841−5.846 21.110 1.00 38.70 A ATOM 291 CB ASN A 107 19.829 −5.623 22.2411.00 37.51 A ATOM 292 CG ASN A 107 18.406 −5.947 21.826 1.00 36.75 AATOM 293 OD1 ASN A 107 17.509 −6.028 22.667 1.00 38.88 A ATOM 294 ND2ASN A 107 18.188 −6.120 20.531 1.00 32.53 A ATOM 295 C ASN A 107 22.168−5.161 21.457 1.00 38.73 A ATOM 296 O ASN A 107 22.425 −4.029 21.0301.00 38.05 A ATOM 297 N ALA A 108 23.010 −5.854 22.224 1.00 37.25 A ATOM298 CA ALA A 108 24.306 −5.319 22.616 1.00 37.11 A ATOM 299 CB ALA A 10824.944 −6.218 23.689 1.00 35.32 A ATOM 300 C ALA A 108 25.208 −5.22021.384 1.00 38.35 A ATOM 301 O ALA A 108 25.949 −4.248 21.219 1.00 36.53A ATOM 302 N SER A 109 25.136 −6.232 20.524 1.00 37.79 A ATOM 303 CA SERA 109 25.918 −6.266 19.290 1.00 39.56 A ATOM 304 CB SER A 109 25.660−7.580 18.544 1.00 40.09 A ATOM 305 OG SER A 109 26.349 −7.615 17.3091.00 43.33 A ATOM 306 C SER A 109 25.542 −5.078 18.397 1.00 38.52 A ATOM307 O SER A 109 26.413 −4.450 17.787 1.00 40.46 A ATOM 308 N ASP A 11024.249 −4.776 18.322 1.00 37.55 A ATOM 309 CA ASP A 110 23.765 −3.64517.523 1.00 38.97 A ATOM 310 CB ASP A 110 22.236 −3.578 17.545 1.0039.76 A ATOM 311 CG ASP A 110 21.580 −4.651 16.686 1.00 42.87 A ATOM 312OD1 ASP A 110 20.344 −4.769 16.743 1.00 41.85 A ATOM 313 OD2 ASP A 11022.290 −5.373 15.956 1.00 46.31 A ATOM 314 C ASP A 110 24.327 −2.32618.060 1.00 36.57 A ATOM 315 O ASP A 110 24.750 −1.469 17.284 1.00 37.95A ATOM 316 N ALA A 111 24.315 −2.173 19.385 1.00 36.03 A ATOM 317 CA ALAA 111 24.827 −0.969 20.047 1.00 35.99 A ATOM 318 CB ALA A 111 24.577−1.047 21.560 1.00 34.35 A ATOM 319 C ALA A 111 26.315 −0.808 19.7791.00 36.64 A ATOM 320 O ALA A 111 26.801 0.310 19.586 1.00 34.89 A ATOM321 N LEU A 112 27.043 −1.926 19.787 1.00 35.89 A ATOM 322 CA LEU A 11228.472 −1.904 19.518 1.00 37.87 A ATOM 323 CB LEU A 112 29.104 −3.26019.871 1.00 37.98 A ATOM 324 CG LEU A 112 29.277 −3.464 21.385 1.0038.68 A ATOM 325 CD1 LEU A 112 29.498 −4.929 21.706 1.00 40.90 A ATOM326 CD2 LEU A 112 30.442 −2.617 21.877 1.00 38.61 A ATOM 327 C LEU A 11228.734 −1.540 18.052 1.00 38.56 A ATOM 328 O LEU A 112 29.636 −0.75217.763 1.00 38.58 A ATOM 329 N ASP A 113 27.944 −2.096 17.132 1.00 38.47A ATOM 330 CA ASP A 113 28.105 −1.775 15.710 1.00 40.19 A ATOM 331 CBASP A 113 27.090 −2.523 14.836 1.00 41.72 A ATOM 332 CG ASP A 113 27.414−3.997 14.665 1.00 44.88 A ATOM 333 OD1 ASP A 113 28.606 −4.350 14.5691.00 46.86 A ATOM 334 OD2 ASP A 113 26.463 −4.804 14.592 1.00 47.83 AATOM 335 C ASP A 113 27.866 −0.279 15.509 1.00 40.20 A ATOM 336 O ASP A113 28.556 0.376 14.724 1.00 40.35 A ATOM 337 N LYS A 114 26.880 0.25116.227 1.00 39.26 A ATOM 338 CA LYS A 114 26.517 1.656 16.109 1.00 41.87A ATOM 339 CB LYS A 114 25.252 1.939 16.927 1.00 45.64 A ATOM 340 CG LYSA 14 24.537 3.228 16.533 1.00 51.03 A ATOM 341 CD LYS A 14 23.068 3.21916.963 1.00 54.21 A ATOM 342 CE LYS A 14 22.332 4.453 16.434 1.00 56.69A ATOM 343 NZ LYS A 14 20.871 4.458 16.760 1.00 58.33 A ATOM 344 C LYS A14 27.648 2.591 16.523 1.00 40.73 A ATOM 345 O LYS A 14 27.992 3.51215.781 1.00 40.09 A ATOM 346 N ILE A 15 28.233 2.361 17.696 1.00 39.71 AATOM 347 CA ILE A 15 29.333 3.214 18.138 1.00 39.52 A ATOM 348 CB ILE A15 29.692 2.974 19.630 1.00 38.85 A ATOM 349 CG2 ILE A 15 30.385 1.61419.809 1.00 39.36 A ATOM 350 CG1 ILE A 15 30.589 4.113 20.133 1.00 40.22A ATOM 351 CD1 ILE A 15 29.897 5.482 20.136 1.00 38.97 A ATOM 352 C ILEA 15 30.561 2.976 17.252 1.00 40.05 A ATOM 353 O ILE A 15 31.352 3.89017.018 1.00 40.80 A ATOM 354 N ARG A 16 30.728 1.761 16.739 1.00 40.94 AATOM 355 CA ARG A 16 31.874 1.517 15.864 1.00 45.55 A ATOM 356 CB ARG A16 32.011 0.038 15.494 1.00 47.82 A ATOM 357 CG ARG A 16 33.361 −0.25914.851 1.00 53.66 A ATOM 358 CD ARG A 16 33.292 −1.334 13.780 1.00 58.47A ATOM 359 NE ARG A 16 33.135 −2.680 14.322 1.00 63.09 A ATOM 360 CZ ARGA 16 33.059 −3.775 13.570 1.00 64.30 A ATOM 361 NH1 ARG A 16 33.128−3.676 12.248 1.00 64.89 A ATOM 362 NH2 ARG A 16 32.914 −4.968 14.1361.00 64.89 A ATOM 363 C ARG A 16 31.728 2.353 14.580 1.00 44.91 A ATOM364 O ARG A 16 32.699 2.927 14.099 1.00 45.70 A ATOM 365 N LEU A 1730.516 2.432 14.039 1.00 45.28 A ATOM 366 CA LEU A 17 30.284 3.22112.832 1.00 45.31 A ATOM 367 CB LEU A 17 28.890 2.944 12.262 1.00 47.48A ATOM 368 CG LEU A 17 28.578 1.500 11.848 1.00 48.18 A ATOM 369 CD1 LEUA 17 27.137 1.415 11.360 1.00 49.37 A ATOM 370 CD2 LEU A 17 29.544 1.04510.760 1.00 50.59 A ATOM 371 C LEU A 17 30.426 4.714 13.144 1.00 45.31 AATOM 372 O LEU A 17 30.942 5.485 12.331 1.00 42.68 A ATOM 373 N ILE A 1829.970 5.125 14.320 1.00 43.80 A ATOM 374 CA ILE A 18 30.083 6.52814.690 1.00 44.18 A ATOM 375 CB ILE A 18 29.377 6.823 16.026 1.00 43.51A ATOM 376 CG2 ILE A 18 29.654 8.267 16.449 1.00 42.87 A ATOM 377 CG1ILE A 18 27.870 6.596 15.878 1.00 43.94 A ATOM 378 CD1 ILE A 18 27.1236.562 17.205 1.00 44.35 A ATOM 379 C ILE A 18 31.552 6.924 14.807 1.0043.76 A ATOM 380 O ILE A 18 31.924 8.055 14.479 1.00 42.32 A ATOM 381 NSER A 19 32.390 5.990 15.257 1.00 44.58 A ATOM 382 CA SER A 19 33.8176.267 15.426 1.00 44.85 A ATOM 383 CB SER A 19 34.538 5.074 16.073 1.0045.06 A ATOM 384 OG SER A 19 34.815 4.048 15.132 1.00 46.75 A ATOM 385 CSER A 19 34.485 6.602 14.092 1.00 45.54 A ATOM 386 O SER A 19 35.5897.140 14.070 1.00 44.02 A ATOM 387 N LEU A 120 33.824 6.268 12.986 1.0045.74 A ATOM 388 CA LEU A 120 34.370 6.559 11.663 1.00 49.20 A ATOM 389CB LEU A 120 33.709 5.678 10.594 1.00 48.81 A ATOM 390 CG LEU A 12033.841 4.154 10.658 1.00 50.97 A ATOM 391 CD1 LEU A 120 32.957 3.5459.586 1.00 51.32 A ATOM 392 CD2 LEU A 120 35.289 3.727 10.460 1.00 51.93A ATOM 393 C LEU A 120 34.145 8.027 11.297 1.00 49.95 A ATOM 394 O LEU A120 34.862 8.578 10.461 1.00 50.69 A ATOM 395 N THR A 121 33.147 8.64811.922 1.00 51.96 A ATOM 396 CA THR A 121 32.809 10.045 11.653 1.0054.50 A ATOM 397 CB THR A 121 31.312 10.212 11.324 1.00 54.43 A ATOM 398OG1 THR A 121 30.526 9.930 12.491 1.00 55.71 A ATOM 399 CG2 THR A 12130.906 9.273 10.203 1.00 54.95 A ATOM 400 C THR A 121 33.123 10.98512.814 1.00 56.48 A ATOM 401 O THR A 121 33.056 12.204 12.663 1.00 57.04A ATOM 402 N ASP A 122 33.451 10.421 13.971 1.00 57.38 A ATOM 403 CA ASPA 122 33.771 11.230 15.143 1.00 58.24 A ATOM 404 CB ASP A 122 32.55211.301 16.070 1.00 59.87 A ATOM 405 CG ASP A 122 32.836 12.036 17.3691.00 61.77 A ATOM 406 OD1 ASP A 122 33.541 13.071 17.343 1.00 62.10 AATOM 407 OD2 ASP A 122 32.337 11.577 18.419 1.00 62.22 A ATOM 408 C ASPA 122 34.978 10.632 15.858 1.00 57.50 A ATOM 409 O ASP A 122 34.8869.570 16.474 1.00 56.22 A ATOM 410 N ALA A 123 36.109 11.328 15.757 1.0056.69 A ATOM 411 CA ALA A 123 37.371 10.892 16.352 1.00 56.90 A ATOM 412CB ALA A 123 38.488 11.853 15.946 1.00 57.71 A ATOM 413 C ALA A 12337.359 10.733 17.870 1.00 57.14 A ATOM 414 O ALA A 123 38.174 9.99418.420 1.00 57.29 A ATOM 415 N ASN A 124 36.454 11.429 18.550 1.00 57.40A ATOM 416 CA ASN A 124 36.370 11.336 20.009 1.00 57.97 A ATOM 417 CBASN A 124 36.222 12.732 20.625 1.00 60.18 A ATOM 418 CG ASN A 124 37.45813.592 20.429 1.00 62.71 A ATOM 419 OD1 ASN A 124 37.791 13.980 19.3081.00 64.70 A ATOM 420 ND2 ASN A 124 38.148 13.890 21.522 1.00 63.57 AATOM 421 C ASN A 124 35.204 10.461 20.466 1.00 56.61 A ATOM 422 O ASN A124 34.810 10.496 21.627 1.00 56.62 A ATOM 423 N ALA A 125 34.664 9.66619.550 1.00 55.85 A ATOM 424 CA ALA A 125 33.533 8.799 19.856 1.00 54.44A ATOM 425 CB ALA A 125 33.096 8.065 18.598 1.00 54.60 A ATOM 426 C ALAA 125 33.774 7.795 20.982 1.00 53.61 A ATOM 427 O ALA A 125 32.885 7.55621.795 1.00 52.10 A ATOM 428 N LEU A 126 34.969 7.213 21.035 1.00 53.75A ATOM 429 CA LEU A 126 35.281 6.210 22.059 1.00 54.69 A ATOM 430 CB LEUA 126 36.083 5.069 21.428 1.00 54.14 A ATOM 431 CG LEU A 126 35.4134.371 20.242 1.00 55.12 A ATOM 432 CD1 LEU A 126 36.371 3.362 19.6251.00 54.89 A ATOM 433 CD2 LEU A 126 34.136 3.687 20.708 1.00 55.04 AATOM 434 C LEU A 126 36.031 6.729 23.285 1.00 54.49 A ATOM 435 O LEU A126 36.515 5.939 24.098 1.00 54.80 A ATOM 436 N ALA A 127 36.111 8.05023.417 1.00 54.59 A ATOM 437 CA ALA A 127 36.813 8.696 24.525 1.00 55.79A ATOM 438 CB ALA A 127 36.791 10.214 24.328 1.00 55.81 A ATOM 439 C ALAA 127 36.283 8.352 25.918 1.00 56.37 A ATOM 440 O ALA A 127 37.032 8.39326.897 1.00 56.95 A ATOM 441 N GLY A 128 34.998 8.024 26.012 1.00 56.51A ATOM 442 CA GLY A 128 34.410 7.695 27.302 1.00 56.45 A ATOM 443 C GLYA 128 34.758 6.303 27.794 1.00 56.02 A ATOM 444 O GLY A 128 34.720 6.03128.992 1.00 56.77 A ATOM 445 N ASN A 129 35.095 5.421 26.861 1.00 56.46A ATOM 446 CA ASN A 129 35.460 4.046 27.175 1.00 56.64 A ATOM 447 CB ASNA 129 34.206 3.251 27.569 1.00 56.17 A ATOM 448 CG ASN A 129 34.5301.877 28.136 1.00 56.13 A ATOM 449 OD1 ASN A 129 33.748 1.311 28.8981.00 56.76 A ATOM 450 ND2 ASN A 129 35.677 1.330 27.755 1.00 54.99 AATOM 451 C ASN A 129 36.093 3.491 25.904 1.00 57.41 A ATOM 452 O ASN A129 35.448 3.416 24.861 1.00 58.38 A ATOM 453 N GLU A 130 37.359 3.10425.991 1.00 58.34 A ATOM 454 CA GLU A 130 38.081 2.605 24.826 1.00 59.34A ATOM 455 CB GLU A 130 39.585 2.730 25.075 1.00 62.83 A ATOM 456 CG GLUA 130 40.046 4.156 25.346 1.00 66.60 A ATOM 457 CD GLU A 130 39.8395.077 24.158 1.00 68.82 A ATOM 458 OE1 GLU A 130 40.121 6.289 24.2861.00 70.09 A ATOM 459 OE2 GLU A 130 39.398 4.590 23.094 1.00 70.56 AATOM 460 C GLU A 130 37.766 1.190 24.353 1.00 57.95 A ATOM 461 O GLU A130 38.109 0.827 23.230 1.00 59.19 A ATOM 462 N ALA A 131 37.112 0.38925.184 1.00 56.39 A ATOM 463 CA ALA A 131 36.802 −0.986 24.795 1.0054.28 A ATOM 464 CB ALA A 131 36.816 −1.888 26.031 1.00 55.68 A ATOM 465C ALA A 131 35.478 −1.160 24.050 1.00 52.74 A ATOM 466 O ALA A 13134.568 −0.340 24.172 1.00 52.73 A ATOM 467 N LEU A 132 35.393 −2.23823.271 1.00 49.97 A ATOM 468 CA LEU A 132 34.193 −2.592 22.511 1.0048.60 A ATOM 469 CB LEU A 132 34.429 −2.403 21.010 1.00 49.50 A ATOM 470CG LEU A 132 34.704 −0.959 20.585 1.00 50.36 A ATOM 471 CD1 LEU A 13235.029 −0.908 19.098 1.00 52.14 A ATOM 472 CD2 LEU A 132 33.491 −0.09120.914 1.00 48.99 A ATOM 473 C LEU A 132 33.927 −4.065 22.822 1.00 48.59A ATOM 474 O LEU A 132 34.354 −4.960 22.082 1.00 46.69 A ATOM 475 N THRA 133 33.217 −4.308 23.922 1.00 45.79 A ATOM 476 CA THR A 133 32.9375.672 24.368 1.00 45.37 A ATOM 477 CB THR A 133 33.929 −6.090 25.4641.00 46.45 A ATOM 478 OG1 THR A 133 33.769 −5.206 26.583 1.00 47.94 AATOM 479 CG2 THR A 133 35.372 −6.010 24.974 1.00 48.42 A ATOM 480 C THRA 133 31.554 −5.867 24.986 1.00 44.45 A ATOM 481 O THR A 133 30.838−4.903 25.279 1.00 41.44 A ATOM 482 N VAL A 134 31.213 −7.138 25.1961.00 43.48 A ATOM 483 CA VAL A 134 29.969 −7.550 25.842 1.00 43.09 AATOM 484 CB VAL A 134 29.083 −8.419 24.926 1.00 43.69 A ATOM 485 CG1 VALA 134 27.858 8.883 25.696 1.00 44.19 A ATOM 486 CG2 VAL A 134 28.652−7.632 23.691 1.00 42.76 A ATOM 487 C VAL A 134 30.424 −8.421 27.0141.00 44.48 A ATOM 488 O VAL A 134 31.087 −9.450 26.812 1.00 43.22 A ATOM489 N LYS A 135 30.081 −8.012 28.231 1.00 43.24 A ATOM 490 CA LYS A 13530.475 −8.756 29.418 1.00 43.72 A ATOM 491 CB LYS A 135 31.421 −7.91230.273 1.00 44.84 A ATOM 492 CG LYS A 135 32.751 −7.639 29.585 1.0046.03 A ATOM 493 CD LYS A 135 33.645 −6.710 30.389 1.00 47.51 A ATOM 494CE LYS A 135 35.024 −6.599 29.741 1.00 48.30 A ATOM 495 NZ LYS A 13535.943 −5.750 30.545 1.00 48.86 A ATOM 496 C LYS A 135 29.269 −9.19030.234 1.00 43.79 A ATOM 497 O LYS A 135 28.378 −8.394 30.525 1.00 41.89A ATOM 498 N ILE A 136 29.251 −10.463 30.603 1.00 44.62 A ATOM 499 CAILE A 136 28.140 −11.008 31.366 1.00 45.53 A ATOM 500 CB ILE A 13627.408 −12.072 30.535 1.00 46.56 A ATOM 501 CG2 ILE A 136 26.293 −12.71731.354 1.00 47.11 A ATOM 502 CG1 ILE A 136 26.849 −11.426 29.264 1.0046.22 A ATOM 503 CD1 ILE A 136 26.351 −12.418 28.240 1.00 49.64 A ATOM504 C ILE A 136 28.601 −11.627 32.678 1.00 46.73 A ATOM 505 O ILE A 13629.626 −12.306 32.726 1.00 46.05 A ATOM 506 N LYS A 137 27.850 −11.37433.744 1.00 47.82 A ATOM 507 CA LYS A 137 28.169 −11.943 35.045 1.0050.85 A ATOM 508 CB LYS A 137 29.052 −10.997 35.871 1.00 53.80 A ATOM509 CO LYS A 137 28.679 −9.522 35.844 1.00 56.43 A ATOM 510 CD LYS A 13729.643 −8.729 36.725 1.00 58.40 A ATOM 511 CE LYS A 137 29.651 −7.23836.394 1.00 60.49 A ATOM 512 NZ LYS A 137 28.329 −6.579 36.598 1.0061.87 A ATOM 513 C LYS A 137 26.924 −12.326 35.838 1.00 51.75 A ATOM 514O LYS A 137 25.897 −11.648 35.791 1.00 49.24 A ATOM 515 N CYS A 13827.028 −13.440 36.553 1.00 52.27 A ATOM 516 CA CYS A 138 25.933 −13.94437.366 1.00 53.85 A ATOM 517 CB CYS A 138 25.862 −15.474 37.269 1.0054.76 A ATOM 518 SG CYS A 138 25.881 −16.147 35.591 1.00 57.29 A ATOM519 C CYS A 138 26.204 −13.551 38.815 1.00 53.98 A ATOM 520 O CYS A 13827.355 −13.475 39.241 1.00 53.90 A ATOM 521 N ASP A 139 25.145 −13.28639.565 1.00 54.37 A ATOM 522 CA ASP A 139 25.290 −12.938 40.971 1.0055.78 A ATOM 523 CB ASP A 139 25.045 −11.446 41.197 1.00 56.31 A ATOM524 CG ASP A 139 25.455 −11.002 42.579 1.00 57.02 A ATOM 525 OD1 ASP A139 25.008 −11.636 43.556 1.00 56.63 A ATOM 526 OD2 ASP A 139 26.227−10.026 42.690 1.00 57.45 A ATOM 527 C ASP A 139 24.258 −13.754 41.7331.00 56.37 A ATOM 528 O ASP A 139 23.184 −13.256 42.079 1.00 56.43 AATOM 529 N ALA A 140 24.586 −15.020 41.974 1.00 56.65 A ATOM 530 CA ALAA 140 23.689 −15.925 42.673 1.00 57.35 A ATOM 531 CB ALA A 140 24.371−17.280 42.879 1.00 57.61 A ATOM 532 C ALA A 140 23.238 −15.353 44.0091.00 57.65 A ATOM 533 O ALA A 140 22.055 −15.380 44.331 1.00 58.06 AATOM 534 N GLU A 141 24.183 −14.825 44.779 1.00 59.43 A ATOM 535 CA GLUA 141 23.875 −14.261 46.088 1.00 60.59 A ATOM 536 CB GLU A 141 25.145−13.691 46.731 1.00 63.85 A ATOM 537 CG GLU A 141 26.252 14.723 46.9381.00 67.61 A ATOM 538 CD GLU A 141 27.452 −14.163 47.687 1.00 69.93 AATOM 539 OE1 GLU A 141 27.928 −13.065 47.323 1.00 72.18 A ATOM 540 OE2GLU A 141 27.926 14.824 48.638 1.00 71.05 A ATOM 541 C GLU A 141 22.795−13.184 46.039 1.00 59.63 A ATOM 542 O GLU A 141 21.989 −13.067 46.9651.00 60.53 A ATOM 543 N ALA A 142 22.772 −12.403 44.962 1.00 56.79 AATOM 544 CA ALA A 142 21.781 −11.339 44.824 1.00 53.70 A ATOM 545 CB ALAA 142 22.471 −10.032 44.453 1.00 53.90 A ATOM 546 C ALA A 142 20.691−11.660 43.803 1.00 51.59 A ATOM 547 O ALA A 142 19.819 −10.834 43.5451.00 52.00 A ATOM 548 N ALA A 143 20.740 −12.859 43.229 1.00 48.22 AATOM 549 CA ALA A 143 19.750 −13.282 42.236 1.00 45.39 A ATOM 550 CB ALAA 143 18.365 −13.366 42.881 1.00 47.05 A ATOM 551 C ALA A 143 19.701−12.352 41.018 1.00 42.69 A ATOM 552 O ALA A 143 18.626 −12.042 40.5091.00 41.09 A ATOM 553 N LEU A 144 20.866 −11.923 40.547 1.00 41.05 AATOM 554 CA LEU A 144 20.936 −11.025 39.397 1.00 41.10 A ATOM 555 CB LEUA 144 21.505 −9.665 39.820 1.00 38.84 A ATOM 556 CG LEU A 144 20.902−8.925 41.015 1.00 40.65 A ATOM 557 CD1 LEU A 144 21.687 −7.632 41.2481.00 41.07 A ATOM 558 CD2 LEU A 144 19.430 −8.629 40.769 1.00 41.05 AATOM 559 C LEU A 144 21.793 −11.560 38.255 1.00 40.77 A ATOM 560 O LEU A144 22.739 12.322 38.470 1.00 42.44 A ATOM 561 N LEU A 145 21.434−11.164 37.038 1.00 39.26 A ATOM 562 CA LEU A 145 22.180 −11.522 35.8381.00 40.45 A ATOM 563 CB LEU A 145 21.353 −12.432 34.924 1.00 41.08 AATOM 564 CG LEU A 145 21.999 12.780 33.571 1.00 44.82 A ATOM 565 CD1 LEUA 145 23.352 13.454 33.801 1.00 44.44 A ATOM 566 CD2 LEU A 145 21.069−13.689 32.761 1.00 44.40 A ATOM 567 C LEU A 145 22.426 −10.170 35.1611.00 39.40 A ATOM 568 O LEU A 145 21.484 9.403 34.958 1.00 38.82 A ATOM569 N HIS A 146 23.682 9.866 34.845 1.00 38.79 A ATOM 570 CA HIS A 14624.022 −8.582 34.212 1.00 38.60 A ATOM 571 CB HIS A 146 25.085 7.81735.015 1.00 37.52 A ATOM 572 CG HIS A 146 24.761 −7.621 36.464 1.0038.83 A ATOM 573 CD2 HIS A 146 25.291 8.184 37.576 1.00 38.65 A ATOM 574ND1 HIS A 146 23.847 −6.688 36.906 1.00 38.20 A ATOM 575 CE1 HIS A 14623.833 −6.681 38.227 1.00 39.86 A ATOM 576 NE2 HIS A 146 24.701 −7.57938.659 1.00 37.50 A ATOM 577 C HIS A 146 24.616 −8.802 32.825 1.00 36.94A ATOM 578 O HIS A 146 25.496 −9.644 32.661 1.00 37.49 A ATOM 579 N VALA 147 24.143 −8.039 31.844 1.00 35.57 A ATOM 580 CA VAL A 147 24.6628.102 30.478 1.00 36.33 A ATOM 581 CB VAL A 147 23.599 8.553 29.478 1.0035.23 A ATOM 582 CG1 VAL A 147 24.188 −8.592 28.082 1.00 37.63 A ATOM583 CG2 VAL A 147 23.096 9.941 29.858 1.00 37.09 A ATOM 584 C VAL A 14725.087 −6.669 30.160 1.00 36.34 A ATOM 585 O VAL A 147 24.250 −5.77829.980 1.00 34.90 A ATOM 586 N THR A 148 26.395 −6.455 30.120 1.00 37.82A ATOM 587 CA THR A 148 26.956 5.131 29.881 1.00 38.60 A ATOM 588 CB THRA 148 27.940 4.750 31.010 1.00 39.96 A ATOM 589 OG1 THR A 148 27.2594.791 32.267 1.00 39.52 A ATOM 590 CG2 THR A 148 28.517 3.339 30.7861.00 41.58 A ATOM 591 C THR A 148 27.702 5.012 28.571 1.00 38.59 A ATOM592 O THR A 148 28.513 −5.873 28.224 1.00 37.69 A ATOM 593 N ASP A 14927.425 −3.938 27.840 1.00 37.49 A ATOM 594 CA ASP A 149 28.112 −3.69426.593 1.00 37.69 A ATOM 595 CB ASP A 149 27.166 −3.865 25.388 1.0038.06 A ATOM 596 CG ASP A 149 26.100 −2.779 25.299 1.00 39.30 A ATOM 597OD1 ASP A 149 26.449 −1.599 25.056 1.00 39.14 A ATOM 598 OD2 ASP A 14924.905 −3.107 25.471 1.00 37.40 A ATOM 599 C ASP A 149 28.687 −2.28626.637 1.00 37.48 A ATOM 600 O ASP A 149 28.236 −1.442 27.412 1.00 37.39A ATOM 601 N THR A 150 29.710 −2.048 25.832 1.00 38.71 A ATOM 602 CA THRA 150 30.323 0.733 25.771 1.00 38.68 A ATOM 603 CB THR A 150 31.8680.834 25.879 1.00 40.35 A ATOM 604 OG1 THR A 150 32.365 1.798 24.9401.00 40.36 A ATOM 605 CG2 THR A 150 32.268 −1.257 27.289 1.00 39.00 AATOM 606 C THR A 150 29.904 0.088 24.449 1.00 39.32 A ATOM 607 O THR A150 30.683 0.617 23.799 1.00 39.69 A ATOM 608 N GLY A 151 28.648 −0.33824.075 1.00 38.91 A ATOM 609 CA GLY A 151 28.089 0.199 22.845 1.00 39.06A ATOM 610 C GLY A 151 27.774 1.683 22.904 1.00 38.81 A ATOM 611 O GLY A151 28.286 2.401 23.764 1.00 37.68 A ATOM 612 N VAL A 152 26.921 2.13921.990 1.00 38.14 A ATOM 613 CA VAL A 152 26.539 3.547 21.916 1.00 36.85A ATOM 614 CB VAL A 152 25.663 3.800 20.660 1.00 36.97 A ATOM 615 CG1VAL A 152 24.359 3.030 20.784 1.00 34.47 A ATOM 616 CG2 VAL A 152 25.3985.312 20.473 1.00 36.38 A ATOM 617 C VAL A 152 25.797 4.071 23.150 1.0038.24 A ATOM 618 O VAL A 152 25.862 5.264 23.457 1.00 37.41 A ATOM 619 NGLY A 153 25.090 3.188 23.854 1.00 35.89 A ATOM 620 CA GLY A 153 24.3443.613 25.023 1.00 33.35 A ATOM 621 C GLY A 153 23.074 4.371 24.641 1.0033.66 A ATOM 622 O GLY A 153 22.769 4.519 23.458 1.00 33.60 A ATOM 623 NMET A 154 22.339 4.844 25.645 1.00 33.73 A ATOM 624 CA MET A 154 21.0985.583 25.435 1.00 35.02 A ATOM 625 CB MET A 154 19.881 4.687 25.713 1.0035.45 A ATOM 626 CG MET A 154 19.672 3.579 24.711 1.00 36.62 A ATOM 627SD MET A 154 18.279 2.497 25.176 1.00 39.95 A ATOM 628 CE MET A 15419.165 1.378 26.258 1.00 38.04 A ATOM 629 C MET A 154 20.991 6.80226.353 1.00 34.74 A ATOM 630 O MET A 154 21.330 6.734 27.532 1.00 34.79A ATOM 631 N THR A 155 20.503 7.907 25.801 1.00 36.95 A ATOM 632 CA THRA 155 20.296 9.127 26.580 1.00 37.93 A ATOM 633 CB THR A 155 20.06010.334 25.670 1.00 35.53 A ATOM 634 OG1 THR A 155 18.860 10.115 24.9171.00 36.10 A ATOM 635 CG2 THR A 155 21.228 10.526 24.714 1.00 34.17 AATOM 636 C THR A 155 19.015 8.932 27.392 1.00 39.62 A ATOM 637 O THR A155 18.270 7.965 27.180 1.00 38.97 A ATOM 638 N ARG A 156 18.738 9.85928.303 1.00 40.35 A ATOM 639 CA ARG A 156 17.531 9.767 29.111 1.00 38.63A ATOM 640 CB ARG A 156 17.416 10.973 30.042 1.00 40.13 A ATOM 641 CGARG A 156 16.189 10.930 30.923 1.00 42.27 A ATOM 642 CD ARG A 156 16.05612.189 31.769 1.00 45.98 A ATOM 643 NE ARG A 156 15.014 12.046 32.7801.00 48.94 A ATOM 644 CZ ARG A 156 13.709 12.121 32.539 1.00 50.12 AATOM 645 NH1 ARG A 156 13.264 12.348 31.312 1.00 50.52 A ATOM 646 NH2ARG A 156 12.846 11.951 33.532 1.00 52.90 A ATOM 647 C ARG A 156 16.2899.688 28.224 1.00 39.01 A ATOM 648 O ARG A 156 15.381 8.874 28.478 1.0036.95 A ATOM 649 N ALA A 157 16.245 10.529 27.185 1.00 35.34 A ATOM 650CA ALA A 157 15.111 10.549 26.267 1.00 35.84 A ATOM 651 CB ALA A 15715.255 11.700 25.252 1.00 36.22 A ATOM 652 C ALA A 157 14.952 9.21625.523 1.00 35.61 A ATOM 653 O ALA A 157 13.839 8.763 25.300 1.00 35.81A ATOM 654 N GLU A 158 16.061 8.597 25.142 1.00 35.85 A ATOM 655 CA GLUA 158 16.003 7.314 24.438 1.00 38.21 A ATOM 656 CB GLU A 158 17.3726.982 23.852 1.00 37.42 A ATOM 657 CG GLU A 158 17.828 8.013 22.815 1.0042.18 A ATOM 658 CD GLU A 158 19.127 7.649 22.141 1.00 41.21 A ATOM 659OE1 GLU A 158 20.131 7.435 22.843 1.00 42.08 A ATOM 660 OE2 GLU A 15819.153 7.587 20.897 1.00 47.22 A ATOM 661 C GLU A 158 15.511 6.17425.346 1.00 37.73 A ATOM 662 O GLU A 158 14.792 5.283 24.895 1.00 38.81A ATOM 663 N LEU A 159 15.896 6.200 26.618 1.00 37.15 A ATOM 664 CA LEUA 159 15.453 5.176 27.561 1.00 37.47 A ATOM 665 CB LEU A 159 16.0585.412 28.949 1.00 37.32 A ATOM 666 CG LEU A 159 17.538 5.060 29.108 1.0038.03 A ATOM 667 CD1 LEU A 159 18.033 5.485 30.493 1.00 38.93 A ATOM 668CD2 LEU A 159 17.727 3.564 28.894 1.00 37.21 A ATOM 669 C LEU A 15913.939 5.216 27.649 1.00 38.76 A ATOM 670 O LEU A 159 13.262 4.17227.614 1.00 37.86 A ATOM 671 N VAL A 160 13.409 6.432 27.766 1.00 36.40A ATOM 672 CA VAL A 160 11.974 6.634 27.839 1.00 36.76 A ATOM 673 CB VALA 160 11.627 8.115 28.159 1.00 36.24 A ATOM 674 CG1 VAL A 160 10.1128.323 28.072 1.00 37.44 A ATOM 675 CG2 VAL A 160 12.131 8.486 29.5461.00 34.35 A ATOM 676 C VAL A 160 11.266 6.248 26.536 1.00 36.92 A ATOM677 O VAL A 160 10.315 5.463 26.548 1.00 37.95 A ATOM 678 N ALA A 16111.730 6.786 25.413 1.00 34.61 A ATOM 679 CA ALA A 161 11.091 6.51624.128 1.00 35.52 A ATOM 680 CB ALA A 161 11.512 7.573 23.110 1.00 35.95A ATOM 681 C ALA A 161 11.304 5.129 23.523 1.00 34.49 A ATOM 682 O ALA A161 10.349 4.476 23.115 1.00 36.23 A ATOM 683 N ASN A 162 12.555 4.69823.457 1.00 34.89 A ATOM 684 CA ASN A 162 12.898 3.422 22.839 1.00 36.90A ATOM 685 CB ASN A 162 14.414 3.331 22.691 1.00 37.47 A ATOM 686 CG ASNA 162 14.961 4.415 21.785 1.00 42.03 A ATOM 687 OD1 ASN A 162 14.3835.505 21.688 1.00 43.34 A ATOM 688 ND2 ASN A 162 16.075 4.134 21.1251.00 40.97 A ATOM 689 C ASN A 162 12.354 2.196 23.551 1.00 37.70 A ATOM690 O ASN A 162 12.039 1.186 22.908 1.00 37.59 A ATOM 691 N LEU A 16312.232 2.277 24.869 1.00 37.09 A ATOM 692 CA LEU A 163 11.722 1.13525.624 1.00 38.10 A ATOM 693 CB LEU A 163 12.587 0.906 26.864 1.00 35.47A ATOM 694 CG LEU A 163 14.093 0.725 26.645 1.00 34.53 A ATOM 695 CD1LEU A 163 14.792 0.602 28.003 1.00 34.16 A ATOM 696 CD2 LEU A 163 14.361−0.506 25.770 1.00 37.46 A ATOM 697 C LEU A 163 10.262 1.297 26.035 1.0038.71 A ATOM 698 O LEU A 163 9.541 0.309 26.209 1.00 38.36 A ATOM 699 NGLY A 164 9.814 2.542 26.163 1.00 37.47 A ATOM 700 CA GLY A 164 8.4552.783 26.602 1.00 36.44 A ATOM 701 C GLY A 164 7.408 3.033 25.541 1.0037.47 A ATOM 702 O GLY A 164 6.217 3.032 25.845 1.00 37.44 A ATOM 703 NTHR A 165 7.838 3.241 24.302 1.00 37.94 A ATOM 704 CA THR A 165 6.9103.508 23.213 1.00 38.31 A ATOM 705 CB THR A 165 6.899 5.002 22.821 1.0039.27 A ATOM 706 OG1 THR A 165 8.014 5.265 21.954 1.00 36.14 A ATOM 707CG2 THR A 165 6.993 5.893 24.058 1.00 39.57 A ATOM 708 C THR A 165 7.3532.775 21.960 1.00 38.40 A ATOM 709 O THR A 165 8.438 2.197 21.906 1.0036.47 A ATOM 710 N ILE A 166 6.505 2.837 20.945 1.00 42.95 A ATOM 711 CAILE A 166 6.811 2.250 19.655 1.00 47.73 A ATOM 712 CB ILE A 166 5.5261.926 18.881 1.00 48.55 A ATOM 713 CG2 ILE A 166 4.566 3.088 18.963 1.0052.33 A ATOM 714 CG1 ILE A 166 5.857 1.594 17.425 1.00 50.22 A ATOM 715CD1 ILE A 166 4.660 1.157 16.620 1.00 50.44 A ATOM 716 C ILE A 166 7.5883.384 18.982 1.00 49.91 A ATOM 717 O ILE A 166 7.013 4.269 18.349 1.0049.34 A ATOM 718 N ALA A 167 8.904 3.353 19.167 1.00 53.09 A ATOM 719 CAALA A 167 9.809 4.375 18.658 1.00 55.59 A ATOM 720 CB ALA A 167 11.1394.277 19.400 1.00 55.40 A ATOM 721 C ALA A 167 10.062 4.404 17.155 1.0057.83 A ATOM 722 O ALA A 167 10.185 5.483 16.575 1.00 57.75 A ATOM 723 NLYS A 168 10.148 3.237 16.522 1.00 61.19 A ATOM 724 CA LYS A 168 10.4183.186 15.085 1.00 63.46 A ATOM 725 CB LYS A 168 11.717 2.421 14.825 1.0064.87 A ATOM 726 CG LYS A 168 12.978 3.188 15.198 1.00 67.57 A ATOM 727CD LYS A 168 13.103 4.488 14.403 1.00 68.54 A ATOM 728 CE LYS A 16813.123 4.234 12.898 1.00 69.62 A ATOM 729 NZ LYS A 168 14.290 3.40912.479 1.00 70.51 A ATOM 730 C LYS A 168 9.318 2.600 14.213 1.00 64.19 AATOM 731 O LYS A 168 8.276 2.159 14.701 1.00 64.53 A ATOM 732 N SER A169 9.571 2.606 12.908 1.00 64.45 A ATOM 733 CA SER A 169 8.632 2.08011.922 1.00 64.31 A ATOM 734 CB SER A 169 9.032 2.537 10.517 1.00 65.39A ATOM 735 OG SER A 169 9.367 3.914 10.499 1.00 69.31 A ATOM 736 C SER A169 8.659 0.558 11.973 1.00 62.99 A ATOM 737 O SER A 169 7.621 −0.09911.892 1.00 62.36 A ATOM 738 N GLY A 170 9.864 0.012 12.105 1.00 62.18 AATOM 739 CA GLY A 170 10.033 −1.427 12.152 1.00 61.11 A ATOM 740 C GLY A170 9.173 −2.103 13.199 1.00 60.68 A ATOM 741 O GLY A 170 8.496 −3.08812.902 1.00 60.52 A ATOM 742 N THR A 171 9.191 −1.575 14.421 1.00 59.30A ATOM 743 CA THR A 171 8.412 −2.158 15.505 1.00 59.38 A ATOM 744 CB THRA 171 8.724 −1.453 16.850 1.00 58.69 A ATOM 745 OG1 THR A 171 7.737−1.810 17.824 1.00 60.23 A ATOM 746 CG2 THR A 171 8.750 0.041 16.6751.00 61.04 A ATOM 747 C THR A 171 6.911 −2.127 15.221 1.00 58.48 A ATOM748 O THR A 171 6.161 −2.973 15.707 1.00 58.04 A ATOM 749 N SER A 1726.479 −1.162 14.415 1.00 58.58 A ATOM 750 CA SER A 172 5.070 −1.04214.056 1.00 58.03 A ATOM 751 CB SER A 172 4.812 0.325 13.407 1.00 59.05A ATOM 752 OG SER A 172 3.428 0.545 13.201 1.00 58.95 A ATOM 753 C SER A172 4.700 −2.174 13.086 1.00 57.97 A ATOM 754 O SER A 172 3.637 −2.78813.205 1.00 57.78 A ATOM 755 N ALA A 173 5.587 −2.447 12.132 1.00 57.28A ATOM 756 CA ALA A 173 5.362 −3.510 11.157 1.00 57.64 A ATOM 757 CB ALAA 173 6.391 −3.424 10.027 1.00 57.77 A ATOM 758 C ALA A 173 5.459 −4.86311.856 1.00 57.34 A ATOM 759 O ALA A 173 4.733 −5.799 11.519 1.00 56.73A ATOM 760 N PHE A 174 6.356 −4.965 12.833 1.00 56.78 A ATOM 761 CA PHEA 174 6.512 −6.212 13.571 1.00 56.41 A ATOM 762 CB PHE A 174 7.542−6.062 14.693 1.00 54.27 A ATOM 763 CG PHE A 174 7.475 −7.157 15.7171.00 51.64 A ATOM 764 CD1 PHE A 174 6.842 −6.947 16.943 1.00 49.88 AATOM 765 CD2 PHE A 174 8.012 −8.416 15.442 1.00 51.59 A ATOM 766 CE1 PHEA 174 6.743 −7.973 17.881 1.00 51.52 A ATOM 767 CE2 PHE A 174 7.920−9.454 16.373 1.00 50.03 A ATOM 768 CZ PHE A 174 7.284 −9.235 17.5951.00 51.03 A ATOM 769 C PHE A 174 5.181 −6.647 14.170 1.00 56.94 A ATOM770 O PHE A 174 4.803 −7.813 14.075 1.00 57.11 A ATOM 771 N LEU A 1754.469 −5.708 14.782 1.00 56.79 A ATOM 772 CA LEU A 175 3.186 −6.02315.395 1.00 58.46 A ATOM 773 CB LEU A 175 2.595 −4.777 16.057 1.00 59.35A ATOM 774 CG LEU A 175 3.267 −4.350 17.364 1.00 61.12 A ATOM 775 CD1LEU A 175 2.691 −3.020 17.834 1.00 61.82 A ATOM 776 CD2 LEU A 175 3.055−5.433 18.419 1.00 62.48 A ATOM 777 C LEU A 175 2.170 −6.623 14.425 1.0058.54 A ATOM 778 O LEU A 175 1.367 −7.476 14.810 1.00 58.31 A ATOM 779 NASN A 176 2.196 −6.177 13.174 1.00 59.10 A ATOM 780 CA ASN A 176 1.263−6.697 12.182 1.00 59.32 A ATOM 781 CB ASN A 176 1.096 −5.711 11.0261.00 61.65 A ATOM 782 CG ASN A 176 0.373 −4.447 11.442 1.00 63.45 A ATOM783 OD1 ASN A 176 −0.610 −4.499 12.188 1.00 64.07 A ATOM 784 ND2 ASN A176 0.844 −3.304 10.954 1.00 63.36 A ATOM 785 C ASN A 176 1.736 −8.03711.641 1.00 58.64 A ATOM 786 O ASN A 176 0.940 −8.962 11.474 1.00 58.77A ATOM 787 N ALA A 177 3.032 −8.136 11.364 1.00 57.09 A ATOM 788 CA ALAA 177 3.600 −9.372 10.846 1.00 56.72 A ATOM 789 CB ALA A 177 5.067−9.164 10.488 1.00 56.36 A ATOM 790 C ALA A 177 3.458 −10.452 11.9071.00 56.28 A ATOM 791 O ALA A 177 3.154 −11.606 11.602 1.00 55.03 A ATOM792 N MET A 178 3.663 10.062 13.162 1.00 56.61 A ATOM 793 CA MET A 1783.560 −10.983 14.287 1.00 58.30 A ATOM 794 CB MET A 178 3.887 10.25615.594 1.00 55.27 A ATOM 795 CG MET A 178 3.969 −11.164 16.797 1.0052.13 A ATOM 796 SD MET A 178 5.240 −12.416 16.581 1.00 49.31 A ATOM 797CE MET A 178 5.117 13.294 18.122 1.00 47.13 A ATOM 798 C MET A 178 2.164−11.587 14.374 1.00 60.81 A ATOM 799 O MET A 178 2.006 −12.810 14.4501.00 61.92 A ATOM 800 N THR A 179 1.151 −10.727 14.356 1.00 62.45 A ATOM801 CA THR A 179 0.233 11.176 14.436 1.00 65.49 A ATOM 802 CB THR A 179−1.198 −9.974 14.570 1.00 66.54 A ATOM 803 OG1 THR A 179 1.012 −9.08313.462 1.00 68.60 A ATOM 804 CG2 THR A 179 −0.936 −9.221 15.865 1.0066.93 A ATOM 805 C THR A 179 0.639 −12.003 13.212 1.00 66.44 A ATOM 806O THR A 179 −1.332 −13.010 13.339 1.00 66.28 A ATOM 807 N GLU A 1800.203 −11.576 12.031 1.00 67.47 A ATOM 808 CA GLU A 180 −0.533 −12.28110.797 1.00 68.76 A ATOM 809 CB GLU A 180 −0.221 −11.404 9.588 1.0070.17 A ATOM 810 CG GLU A 180 −1.273 −10.346 9.302 1.00 72.96 A ATOM 811CD GLU A 180 −0.925 −9.502 8.091 1.00 74.23 A ATOM 812 OE1 GLU A 180−1.792 −8.727 7.629 1.00 74.78 A ATOM 813 OE2 GLU A 180 0.222 9.6137.605 1.00 75.19 A ATOM 814 C GLU A 180 0.195 −13.612 10.654 1.00 69.31A ATOM 815 O GLU A 180 −0.123 14.411 9.773 1.00 69.53 A ATOM 816 N ALA A181 1.175 13.844 11.519 1.00 69.22 A ATOM 817 CA ALA A 181 1.937 −15.08311.488 1.00 69.33 A ATOM 818 CB ALA A 181 3.418 −14.790 11.667 1.0068.64 A ATOM 819 C ALA A 181 1.448 15.984 12.608 1.00 69.85 A ATOM 820 OALA A 181 1.150 −17.159 12.399 1.00 69.63 A ATOM 821 N GLN A 182 1.363−15.410 13.801 1.00 70.54 A ATOM 822 CA GLN A 182 0.926 −16.131 14.9881.00 71.47 A ATOM 823 CB GLN A 182 1.080 −15.218 16.208 1.00 71.17 AATOM 824 CG GLN A 182 0.727 −15.838 17.541 1.00 71.33 A ATOM 825 CD GLNA 182 1.202 −14.988 18.707 1.00 71.75 A ATOM 826 OE1 GLN A 182 2.396−14.928 19.000 1.00 71.32 A ATOM 827 NE2 GLN A 182 0.268 −14.314 19.3691.00 72.54 A ATOM 828 C GLN A 182 −0.516 −16.608 14.851 1.00 71.82 AATOM 829 O GLN A 182 −0.973 −17.462 15.613 1.00 71.86 A ATOM 830 N GLU A183 −1.223 −16.063 13.866 1.00 72.03 A ATOM 831 CA GLU A 183 −2.614−16.426 13.635 1.00 72.86 A ATOM 832 CB GLU A 183 −3.348 −15.277 12.9321.00 74.17 A ATOM 833 CG GLU A 183 −4.826 −15.540 12.667 1.00 76.24 AATOM 834 CD GLU A 183 −5.548 −14.326 12.106 1.00 77.73 A ATOM 835 OE1GLU A 183 −6.730 −14.460 11.716 1.00 78.21 A ATOM 836 OE2 GLU A 183−4.936 −13.235 12.061 1.00 78.20 A ATOM 837 C GLU A 183 −2.758 −17.71012.822 1.00 72.67 A ATOM 838 O GLU A 183 −3.694 −18.481 13.039 1.0072.75 A ATOM 839 N ASP A 184 −1.835 −17.948 11.893 1.00 72.01 A ATOM 840CA ASP A 184 −1.909 −19.151 11.068 1.00 71.44 A ATOM 841 CB ASP A 184−2.078 −18.772 9.592 1.00 73.01 A ATOM 842 CG ASP A 184 −0.847 −18.1139.014 1.00 74.32 A ATOM 843 OD1 ASP A 184 −0.483 −17.016 9.484 1.0076.20 A ATOM 844 OD2 ASP A 184 −0.244 −18.697 8.088 1.00 75.11 A ATOM845 C ASP A 184 −0.726 −20.111 11.230 1.00 69.97 A ATOM 846 O ASP A 184−0.269 −20.725 10.263 1.00 69.71 A ATOM 847 N GLY A 185 −0.235 −20.22612.460 1.00 67.94 A ATOM 848 CA GLY A 185 0.858 −21.136 12.761 1.0065.21 A ATOM 849 C GLY A 185 2.221 −20.990 12.101 1.00 63.26 A ATOM 850O GLY A 185 2.910 −21.995 11.914 1.00 62.89 A ATOM 851 N GLN A 186 2.624−19.774 11.739 1.00 59.88 A ATOM 852 CA GLN A 186 3.945 −19.581 11.1371.00 56.96 A ATOM 853 CB GLN A 186 3.924 −18.417 10.145 1.00 57.15 AATOM 854 CG GLN A 186 3.203 −18.723 8.843 1.00 58.88 A ATOM 855 CD GLN A186 3.159 −17.531 7.905 1.00 59.29 A ATOM 856 OE1 GLN A 186 4.195−16.975 7.538 1.00 61.02 A ATOM 857 NE2 GLN A 186 1.955 −17.132 7.5121.00 59.93 A ATOM 858 C GLN A 186 4.926 −19.298 12.276 1.00 54.13 A ATOM859 O GLN A 186 4.524 −18.755 13.306 1.00 54.33 A ATOM 860 N SER A 1876.195 −19.670 12.107 1.00 49.48 A ATOM 861 CA SER A 187 7.189 −19.45513.162 1.00 47.88 A ATOM 862 CB SER A 187 8.553 −20.008 12.751 1.0046.11 A ATOM 863 OG SER A 187 9.389 −20.159 13.889 1.00 46.52 A ATOM 864C SER A 187 7.312 −17.968 13.495 1.00 46.56 A ATOM 865 O SER A 187 7.303−17.122 12.604 1.00 45.29 A ATOM 866 N THR A 188 7.452 −17.665 14.7791.00 46.91 A ATOM 867 CA THR A 188 7.523 −16.279 15.237 1.00 46.94 AATOM 868 CB THR A 188 6.366 −15.992 16.211 1.00 48.10 A ATOM 869 OG1 THRA 188 6.361 −16.989 17.240 1.00 47.90 A ATOM 870 CG2 THR A 188 5.029−16.020 15.482 1.00 48.15 A ATOM 871 C THR A 188 8.816 −15.810 15.9051.00 46.41 A ATOM 872 O THR A 188 9.011 −14.599 16.084 1.00 45.96 A ATOM873 N SER A 189 9.691 −16.735 16.290 1.00 43.65 A ATOM 874 CA SER A 18910.940 −16.336 16.940 1.00 43.00 A ATOM 875 CB SER A 189 11.678 −17.55517.525 1.00 44.76 A ATOM 876 OG SER A 189 12.138 −18.428 16.505 1.0045.08 A ATOM 877 C SER A 189 11.854 −15.600 15.964 1.00 43.67 A ATOM 878O SER A 189 12.667 −14.760 16.373 1.00 41.00 A ATOM 879 N ALA A 19011.721 −15.908 14.673 1.00 41.06 A ATOM 880 CA ALA A 190 12.542 −15.25513.657 1.00 41.18 A ATOM 881 CB ALA A 190 12.401 −15.967 12.315 1.0042.02 A ATOM 882 C ALA A 190 12.111 −13.798 13.518 1.00 42.09 A ATOM 883O ALA A 190 12.938 −12.919 13.278 1.00 38.77 A ATOM 884 N LEU A 19110.814 −13.555 13.668 1.00 42.41 A ATOM 885 CA LEU A 191 10.281 −12.20413.560 1.00 44.99 A ATOM 886 CB LEU A 191 8.751 −12.219 13.583 1.0044.92 A ATOM 887 CG LEU A 191 8.003 −12.719 12.346 1.00 46.34 A ATOM 888CD1 LEU A 191 6.507 −12.683 12.630 1.00 46.59 A ATOM 889 CD2 LEU A 1918.335 −11.850 11.142 1.00 46.99 A ATOM 890 C LEU A 191 10.789 −11.29614.680 1.00 45.19 A ATOM 891 O LEU A 191 11.202 −10.155 14.423 1.0045.12 A ATOM 892 N ILE A 192 10.759 −11.790 15.915 1.00 44.61 A ATOM 893CA ILE A 192 11.219 −10.979 17.036 1.00 46.52 A ATOM 894 CB ILE A 19211.044 −11.699 18.409 1.00 47.46 A ATOM 895 CG2 ILE A 192 9.594 −12.16218.583 1.00 46.74 A ATOM 896 CG1 ILE A 192 12.002 −12.880 18.513 1.0051.59 A ATOM 897 CD1 ILE A 192 12.338 −13.257 19.948 1.00 55.04 A ATOM898 C ILE A 192 12.688 −10.604 16.842 1.00 45.97 A ATOM 899 O ILE A 19213.085 −9.476 17.118 1.00 44.40 A ATOM 900 N GLY A 193 13.484 −11.55116.351 1.00 45.58 A ATOM 901 CA GLY A 193 14.893 −11.287 16.117 1.0045.18 A ATOM 902 C GLY A 193 15.117 −10.342 14.946 1.00 45.44 A ATOM 903O GLY A 193 15.962 −9.446 15.018 1.00 45.12 A ATOM 904 N GLN A 19414.358 −10.542 13.869 1.00 44.42 A ATOM 905 CA GLN A 194 14.472 −9.70912.672 1.00 46.80 A ATOM 906 CB GLN A 194 13.493 −10.188 11.594 1.0048.06 A ATOM 907 CG GLN A 194 13.938 −11.435 10.851 1.00 52.72 A ATOM908 CD GLN A 194 13.004 −11.798 9.701 1.00 54.82 A ATOM 909 OE1 GLN A194 13.268 −12.739 8.951 1.00 58.37 A ATOM 910 NE2 GLN A 194 11.909−11.054 9.560 1.00 53.81 A ATOM 911 C GLN A 194 14.212 −8.228 12.9471.00 46.26 A ATOM 912 O GLN A 194 14.953 −7.359 12.483 1.00 45.58 A ATOM913 N PHE A 195 13.149 −7.948 13.689 1.00 45.59 A ATOM 914 CA PHE A 19512.785 −6.573 14.012 1.00 47.81 A ATOM 915 CB PHE A 195 11.262 −6.47014.112 1.00 48.72 A ATOM 916 CG PHE A 195 10.571 −6.578 12.783 1.0052.11 A ATOM 917 CD1 PHE A 195 10.450 −5.463 11.955 1.00 53.36 A ATOM918 CD2 PHE A 195 10.084 −7.806 12.333 1.00 53.76 A ATOM 919 CE1 PHE A195 9.855 −5.565 10.693 1.00 54.61 A ATOM 920 CE2 PHE A 195 9.485 −7.92511.072 1.00 54.49 A ATOM 921 CZ PHE A 195 9.371 −6.800 10.249 1.00 55.89A ATOM 922 C PHE A 195 13.452 −6.043 15.281 1.00 45.89 A ATOM 923 O PHEA 195 13.224 −4.898 15.678 1.00 46.00 A ATOM 924 N GLY A 196 14.281−6.880 15.898 1.00 43.45 A ATOM 925 CA GLY A 196 14.991 −6.496 17.1051.00 43.03 A ATOM 926 C GLY A 196 14.139 −6.021 18.269 1.00 41.78 A ATOM927 O GLY A 196 14.488 −5.043 18.935 1.00 39.12 A ATOM 928 N VAL A 19713.032 −6.717 18.529 1.00 39.22 A ATOM 929 CA VAL A 197 12.139 −6.35319.629 1.00 37.80 A ATOM 930 CB VAL A 197 10.659 −6.282 19.155 1.0037.78 A ATOM 931 CG1 VAL A 197 10.498 −5.196 18.101 1.00 39.35 A ATOM932 CG2 VAL A 197 10.212 −7.633 18.592 1.00 39.35 A ATOM 933 C VAL A 19712.228 −7.321 20.821 1.00 36.08 A ATOM 934 O VAL A 197 11.379 −7.29221.708 1.00 33.97 A ATOM 935 N GLY A 198 13.264 −8.159 20.838 1.00 35.72A ATOM 936 CA GLY A 198 13.445 −9.133 21.910 1.00 34.04 A ATOM 937 C GLYA 198 13.722 −8.639 23.324 1.00 35.00 A ATOM 938 O GLY A 198 13.688−9.427 24.269 1.00 32.54 A ATOM 939 N PHE A 199 14.009 −7.349 23.5001.00 34.83 A ATOM 940 CA PHE A 199 14.267 −6.841 24.841 1.00 33.95 AATOM 941 CB PHE A 199 14.400 −5.308 24.804 1.00 35.31 A ATOM 942 CG PHEA 199 14.375 −4.663 26.159 1.00 35.09 A ATOM 943 CD1 PHE A 199 15.521−4.614 26.946 1.00 34.35 A ATOM 944 CD2 PHE A 199 13.183 −4.130 26.6601.00 35.32 A ATOM 945 CE1 PHE A 199 15.490 −4.044 28.225 1.00 34.68 AATOM 946 CE2 PHE A 199 13.131 −3.561 27.929 1.00 34.37 A ATOM 947 CZ PHEA 199 14.295 −3.518 28.720 1.00 35.36 A ATOM 948 C PHE A 199 13.165−7.250 25.829 1.00 33.31 A ATOM 949 O PHE A 199 13.449 −7.652 26.9611.00 31.85 A ATOM 950 N TYR A 200 11.913 −7.178 25.395 1.00 33.78 A ATOM951 CA TYR A 200 10.799 −7.515 26.270 1.00 35.65 A ATOM 952 CB TYR A 2009.479 −7.090 25.621 1.00 35.72 A ATOM 953 CG TYR A 200 9.432 −5.61025.293 1.00 36.99 A ATOM 954 CD1 TYR A 200 9.404 −4.645 26.304 1.0035.32 A ATOM 955 CE1 TYR A 200 9.413 −3.267 25.990 1.00 37.74 A ATOM 956CD2 TYR A 200 9.465 −5.177 23.971 1.00 38.74 A ATOM 957 CE2 TYR A 2009.474 −3.826 23.654 1.00 40.33 A ATOM 958 CZ TYR A 200 9.449 −2.87724.665 1.00 38.60 A ATOM 959 OH TYR A 200 9.463 −1.542 24.326 1.00 43.22A ATOM 960 C TYR A 200 10.716 −8.982 26.707 1.00 35.83 A ATOM 961 O TYRA 200 10.067 −9.284 27.712 1.00 32.96 A ATOM 962 N SER A 201 11.379−9.880 25.978 1.00 35.53 A ATOM 963 CA SER A 201 11.332 −11.301 26.3451.00 36.28 A ATOM 964 CB SER A 201 11.969 −12.185 25.257 1.00 34.75 AATOM 965 OG SER A 201 13.364 −11.984 25.134 1.00 35.49 A ATOM 966 C SERA 201 12.022 −11.544 27.682 1.00 36.13 A ATOM 967 O SER A 201 11.854−12.604 28.299 1.00 36.56 A ATOM 968 N ALA A 202 12.805 −10.563 28.1311.00 35.31 A ATOM 969 CA ALA A 202 13.490 −10.680 29.404 1.00 33.89 AATOM 970 CB ALA A 202 14.315 −9.413 29.692 1.00 36.62 A ATOM 971 C ALA A202 12.495 −10.909 30.527 1.00 32.57 A ATOM 972 O ALA A 202 12.838−11.516 31.546 1.00 33.66 A ATOM 973 N PHE A 203 11.274 −10.409 30.3611.00 30.62 A ATOM 974 CA PHE A 203 10.263 −10.574 31.396 1.00 32.83 AATOM 975 CB PHE A 203 9.056 −9.656 31.144 1.00 34.52 A ATOM 976 CG PHE A203 9.312 −8.195 31.498 1.00 37.83 A ATOM 977 CD1 PHE A 203 9.629 −7.82532.808 1.00 36.70 A ATOM 978 CD2 PHE A 203 9.241 −7.205 30.521 1.0037.21 A ATOM 979 CE1 PHE A 203 9.878 −6.478 33.144 1.00 39.70 A ATOM 980CE2 PHE A 203 9.485 −5.853 30.841 1.00 39.06 A ATOM 981 CZ PHE A 2039.807 −5.492 32.156 1.00 35.63 A ATOM 982 C PHE A 203 9.820 −12.03331.524 1.00 33.89 A ATOM 983 O PHE A 203 9.065 −12.369 32.428 1.00 33.98A ATOM 984 N LEU A 204 10.296 −12.894 30.623 1.00 35.14 A ATOM 985 CALEU A 204 9.964 −14.324 30.710 1.00 37.59 A ATOM 986 CB LEU A 204 10.352−15.061 29.424 1.00 37.82 A ATOM 987 CG LEU A 204 9.396 −14.947 28.2401.00 37.22 A ATOM 988 CD1 LEU A 204 10.077 −15.471 26.997 1.00 41.61 AATOM 989 CD2 LEU A 204 8.113 −15.715 28.533 1.00 38.03 A ATOM 990 C LEUA 204 10.744 −14.922 31.875 1.00 37.39 A ATOM 991 O LEU A 204 10.290−15.867 32.516 1.00 38.71 A ATOM 992 N VAL A 205 11.918 −14.357 32.1621.00 36.51 A ATOM 993 CA VAL A 205 12.744 −14.863 33.248 1.00 36.53 AATOM 994 CB VAL A 205 14.147 −15.325 32.736 1.00 37.09 A ATOM 995 CG1VAL A 205 13.984 −16.446 31.708 1.00 35.55 A ATOM 996 CG2 VAL A 20514.918 −14.146 32.108 1.00 34.89 A ATOM 997 C VAL A 205 12.960 −13.88334.396 1.00 35.28 A ATOM 998 O VAL A 205 13.476 −14.265 35.440 1.0034.02 A ATOM 999 N ALA A 206 12.563 −12.627 34.214 1.00 36.36 A ATOM1000 CA ALA A 206 12.775 −11.635 35.261 1.00 35.50 A ATOM 1001 CB ALA A206 13.787 −10.567 34.773 1.00 35.42 A ATOM 1002 C ALA A 206 11.521−10.945 35.783 1.00 34.37 A ATOM 1003 O ALA A 206 10.611 −10.598 35.0211.00 35.56 A ATOM 1004 N ASP A 207 11.497 −10.758 37.099 1.00 33.09 AATOM 1005 CA ASP A 207 10.411 −10.065 37.791 1.00 33.92 A ATOM 1006 CBASP A 207 10.434 −10.442 39.268 1.00 35.69 A ATOM 1007 CG ASP A 2079.768 −11.784 39.545 1.00 37.34 A ATOM 1008 OD1 ASP A 207 10.273 −12.52140.418 1.00 40.55 A ATOM 1009 OD2 ASP A 207 8.739 −12.085 38.908 1.0036.54 A ATOM 1010 C ASP A 207 10.635 −8.548 37.655 1.00 32.95 A ATOM1011 O ASP A 207 9.698 −7.749 37.764 1.00 36.47 A ATOM 1012 N LYS A 20811.889 −8.180 37.428 1.00 33.18 A ATOM 1013 CA LYS A 208 12.292 −6.78237.261 1.00 35.39 A ATOM 1014 CB LYS A 208 12.688 −6.156 38.611 1.0036.31 A ATOM 1015 CG LYS A 208 13.332 −4.759 38.481 1.00 41.32 A ATOM1016 CD LYS A 208 12.372 −3.622 38.833 1.00 44.97 A ATOM 1017 CE LYS A208 12.173 −3.496 40.338 1.00 46.96 A ATOM 1018 NZ LYS A 208 11.480−2.222 40.715 1.00 48.18 A ATOM 1019 C LYS A 208 13.471 −6.682 36.3011.00 32.95 A ATOM 1020 O LYS A 208 14.414 −7.472 36.353 1.00 31.45 AATOM 1021 N VAL A 209 13.408 −5.693 35.420 1.00 31.85 A ATOM 1022 CA VALA 209 14.469 −5.468 34.461 1.00 30.22 A ATOM 1023 CB VAL A 209 13.948−5.621 33.018 1.00 30.60 A ATOM 1024 CG1 VAL A 209 15.079 −5.326 32.0161.00 32.72 A ATOM 1025 CG2 VAL A 209 13.393 −7.061 32.816 1.00 31.30 AATOM 1026 C VAL A 209 14.967 −4.045 34.691 1.00 33.58 A ATOM 1027 O VALA 209 14.170 −3.104 34.743 1.00 31.38 A ATOM 1028 N ILE A 210 16.274−3.910 34.863 1.00 33.27 A ATOM 1029 CA ILE A 210 16.889 −2.612 35.0861.00 37.08 A ATOM 1030 CB ILE A 210 17.563 −2.558 36.478 1.00 38.79 AATOM 1031 CG2 ILE A 210 18.181 −1.181 36.715 1.00 39.96 A ATOM 1032 CG1ILE A 210 16.521 −2.856 37.557 1.00 37.52 A ATOM 1033 CD1 ILE A 21017.077 −2.837 38.964 1.00 42.42 A ATOM 1034 C ILE A 210 17.915 −2.35333.990 1.00 37.29 A ATOM 1035 O ILE A 210 18.724 −3.228 33.650 1.0036.88 A ATOM 1036 N VAL A 211 17.875 −1.148 33.430 1.00 34.49 A ATOM1037 CA VAL A 211 18.791 −0.793 32.366 1.00 32.75 A ATOM 1038 CB VAL A211 18.038 −0.519 31.036 1.00 33.29 A ATOM 1039 CG1 VAL A 211 19.032−0.186 29.942 1.00 32.50 A ATOM 1040 CG2 VAL A 211 17.211 −1.759 30.6081.00 32.87 A ATOM 1041 C VAL A 211 19.558 0.456 32.786 1.00 34.30 A ATOM1042 O VAL A 211 18.958 1.504 33.015 1.00 31.53 A ATOM 1043 N THR A 21220.870 0.318 32.937 1.00 34.12 A ATOM 1044 CA THR A 212 21.709 1.45733.311 1.00 36.70 A ATOM 1045 CB THR A 212 22.734 1.060 34.372 1.0038.16 A ATOM 1046 OG1 THR A 212 23.592 0.039 33.844 1.00 47.00 A ATOM1047 CG2 THR A 212 22.030 0.527 35.617 1.00 35.54 A ATOM 1048 C THR A212 22.404 1.830 32.017 1.00 35.20 A ATOM 1049 O THR A 212 22.942 0.95631.324 1.00 33.56 A ATOM 1050 N SER A 213 22.395 3.116 31.671 1.00 33.86A ATOM 1051 CA SER A 213 22.980 3.500 30.403 1.00 32.52 A ATOM 1052 CBSER A 213 21.891 3.448 29.328 1.00 29.90 A ATOM 1053 OG SER A 213 22.4653.348 28.047 1.00 31.83 A ATOM 1054 C SER A 213 23.671 4.859 30.364 1.0032.91 A ATOM 1055 O SER A 213 23.199 5.825 30.966 1.00 34.92 A ATOM 1056N LYS A 214 24.776 4.912 29.627 1.00 34.54 A ATOM 1057 CA LYS A 21425.560 6.143 29.462 1.00 35.37 A ATOM 1058 CB LYS A 214 26.862 6.05630.260 1.00 35.40 A ATOM 1059 CG LYS A 214 27.799 7.265 30.062 1.0036.79 A ATOM 1060 CD LYS A 214 27.173 8.547 30.588 1.00 35.02 A ATOM1061 CE LYS A 214 28.148 9.733 30.441 1.00 39.66 A ATOM 1062 NZ LYS A214 28.384 10.085 29.003 1.00 39.15 A ATOM 1063 C LYS A 214 25.884 6.40227.990 1.00 35.51 A ATOM 1064 O LYS A 214 26.663 5.674 27.374 1.00 37.49A ATOM 1065 N HIS A 215 25.272 7.440 27.430 1.00 36.90 A ATOM 1066 CAHIS A 215 25.492 7.828 26.034 1.00 38.06 A ATOM 1067 CB HIS A 215 24.1618.240 25.403 1.00 39.15 A ATOM 1068 CG HIS A 215 24.256 8.634 23.9611.00 42.53 A ATOM 1069 CD2 HIS A 215 24.657 9.789 23.377 1.00 42.04 AATOM 1070 ND1 HIS A 215 23.861 7.802 22.934 1.00 44.41 A ATOM 1071 CE1HIS A 215 24.008 8.429 21.780 1.00 44.09 A ATOM 1072 NE2 HIS A 21524.490 9.638 22.021 1.00 45.63 A ATOM 1073 C HIS A 215 26.439 9.03326.062 1.00 39.69 A ATOM 1074 O HIS A 215 26.408 9.807 27.016 1.00 40.24A ATOM 1075 N ASN A 216 27.263 9.187 25.025 1.00 40.51 A ATOM 1076 CAASN A 216 28.204 10.306 24.935 1.00 42.83 A ATOM 1077 CB ASN A 21628.959 10.291 23.596 1.00 42.89 A ATOM 1078 CG ASN A 216 30.045 9.23323.535 1.00 42.90 A ATOM 1079 OD1 ASN A 216 30.499 8.722 24.556 1.0044.23 A ATOM 1080 ND2 ASN A 216 30.484 8.914 22.323 1.00 43.07 A ATOM1081 C ASN A 216 27.538 11.675 25.080 1.00 42.65 A ATOM 1082 O ASN A 21628.179 12.632 25.519 1.00 44.85 A ATOM 1083 N ASN A 217 26.270 11.78424.701 1.00 42.59 A ATOM 1084 CA ASN A 217 25.567 13.067 24.782 1.0043.43 A ATOM 1085 CB ASN A 217 24.719 13.297 23.523 1.00 45.33 A ATOM1086 CG ASN A 217 25.558 13.388 22.257 1.00 46.89 A ATOM 1087 OD1 ASN A217 26.711 13.826 22.291 1.00 49.15 A ATOM 1088 ND2 ASN A 217 24.97612.986 21.131 1.00 47.14 A ATOM 1089 C ASN A 217 24.679 13.267 26.0031.00 43.78 A ATOM 1090 O ASN A 217 23.786 14.110 25.980 1.00 43.62 AATOM 1091 N ASP A 218 24.915 12.513 27.073 1.00 43.35 A ATOM 1092 CA ASPA 218 24.086 12.662 28.263 1.00 43.44 A ATOM 1093 CB ASP A 218 22.72111.989 28.032 1.00 45.80 A ATOM 1094 CG ASP A 218 21.613 12.575 28.9071.00 47.88 A ATOM 1095 OD1 ASP A 218 21.920 13.368 29.823 1.00 48.42 AATOM 1096 OD2 ASP A 218 20.430 12.237 28.679 1.00 46.04 A ATOM 1097 CASP A 218 24.777 12.033 29.465 1.00 41.31 A ATOM 1098 O ASP A 218 25.81511.385 29.335 1.00 41.97 A ATOM 1099 N THR A 219 24.211 12.244 30.6441.00 41.11 A ATOM 1100 CA THR A 219 24.771 11.658 31.847 1.00 40.99 AATOM 1101 CB THR A 219 24.389 12.487 33.081 1.00 42.30 A ATOM 1102 OG1THR A 219 22.980 12.744 33.058 1.00 43.10 A ATOM 1103 CG2 THR A 21925.150 13.836 33.078 1.00 44.68 A ATOM 1104 C THR A 219 24.188 10.24331.960 1.00 40.58 A ATOM 1105 O THR A 219 23.305 9.868 31.185 1.00 40.58A ATOM 1106 N GLN A 220 24.689 9.463 32.908 1.00 40.36 A ATOM 1107 CAGLN A 220 24.211 8.092 33.102 1.00 39.83 A ATOM 1108 CB GLN A 220 25.2487.299 33.900 1.00 40.67 A ATOM 1109 CG GLN A 220 24.877 5.857 34.2461.00 41.43 A ATOM 1110 CD GLN A 220 26.102 5.047 34.647 1.00 42.56 AATOM 1111 OE1 GLN A 220 26.056 4.239 35.578 1.00 44.52 A ATOM 1112 NE2GLN A 220 27.204 5.252 33.935 1.00 41.54 A ATOM 1113 C GLN A 220 22.8588.062 33.806 1.00 38.08 A ATOM 1114 O GLN A 220 22.641 8.747 34.812 1.0035.10 A ATOM 1115 N HIS A 221 21.947 7.246 33.275 1.00 36.61 A ATOM 1116CA HIS A 221 20.613 7.117 33.833 1.00 34.35 A ATOM 1117 CB HIS A 22119.587 7.755 32.888 1.00 37.28 A ATOM 1118 CG HIS A 221 19.687 9.24932.799 1.00 39.74 A ATOM 1119 CD2 HIS A 221 20.550 10.047 32.127 1.0038.57 A ATOM 1120 ND1 HIS A 221 18.830 10.095 33.471 1.00 41.09 A ATOM1121 CE1 HIS A 221 19.162 11.350 33.218 1.00 39.34 A ATOM 1122 NE2 HIS A221 20.201 11.348 32.405 1.00 40.70 A ATOM 1123 C HIS A 221 20.260 5.63834.044 1.00 35.15 A ATOM 1124 O HIS A 221 20.942 4.742 33.522 1.00 30.35A ATOM 1125 N ILE A 222 19.211 5.416 34.829 1.00 34.92 A ATOM 1126 CAILE A 222 18.706 4.071 35.128 1.00 35.77 A ATOM 1127 CB ILE A 222 18.8513.715 36.638 1.00 34.59 A ATOM 1128 CG2 ILE A 222 18.362 2.274 36.8941.00 36.24 A ATOM 1129 CG1 ILE A 222 20.304 3.849 37.068 1.00 33.15 AATOM 1130 CD1 ILE A 222 20.521 3.735 38.560 1.00 33.87 A ATOM 1131 C ILEA 222 17.218 3.989 34.774 1.00 34.51 A ATOM 1132 O ILE A 222 16.4324.867 35.146 1.00 35.84 A ATOM 1133 N TRP A 223 16.848 2.945 34.029 1.0034.41 A ATOM 1134 CA TRP A 223 15.462 2.679 33.643 1.00 33.72 A ATOM1135 CB TRP A 223 15.391 2.404 32.130 1.00 34.35 A ATOM 1136 CG TRP A223 14.032 2.052 31.545 1.00 34.80 A ATOM 1137 CD2 TRP A 223 13.4440.743 31.460 1.00 33.67 A ATOM 1138 CE2 TRP A 223 12.243 0.868 30.7261.00 34.03 A ATOM 1139 CE3 TRP A 223 13.825 −0.526 31.926 1.00 35.44 AATOM 1140 CD1 TRP A 223 13.173 2.897 30.890 1.00 33.40 A ATOM 1141 NE1TRP A 223 12.096 2.190 30.393 1.00 34.12 A ATOM 1142 CZ2 TRP A 22311.414 −0.234 30.448 1.00 33.49 A ATOM 1143 CZ3 TRP A 223 12.992 −1.62731.645 1.00 32.64 A ATOM 1144 CH2 TRP A 223 11.813 −1.468 30.914 1.0031.20 A ATOM 1145 C TRP A 223 15.150 1.406 34.438 1.00 34.21 A ATOM 1146O TRP A 223 16.015 0.537 34.559 1.00 34.33 A ATOM 1147 N GLU A 22413.956 1.306 35.008 1.00 35.10 A ATOM 1148 CA GLU A 224 13.592 0.11435.780 1.00 36.56 A ATOM 1149 CB GLU A 224 13.952 0.308 37.263 1.0038.53 A ATOM 1150 CG GLU A 224 13.255 1.465 37.945 1.00 40.52 A ATOM1151 CD GLU A 224 11.948 1.052 38.592 1.00 45.21 A ATOM 1152 OE1 GLU A224 11.152 1.951 38.931 1.00 45.71 A ATOM 1153 OE2 GLU A 224 11.723−0.169 38.776 1.00 44.23 A ATOM 1154 C GLU A 224 12.106 −0.196 35.6081.00 35.60 A ATOM 1155 O GLU A 224 11.263 0.708 35.554 1.00 35.06 A ATOM1156 N SER A 225 11.773 −1.481 35.507 1.00 33.72 A ATOM 1157 CA SER A225 10.390 −1.846 35.300 1.00 31.46 A ATOM 1158 CB SER A 225 10.022−1.636 33.830 1.00 33.63 A ATOM 1159 OG SER A 225 8.750 −2.203 33.5411.00 34.39 A ATOM 1160 C SER A 225 10.074 −3.290 35.665 1.00 33.06 AATOM 1161 O SER A 225 10.934 −4.163 35.538 1.00 29.51 A ATOM 1162 N ASPA 226 8.845 −3.515 36.116 1.00 34.95 A ATOM 1163 CA ASP A 226 8.368−4.858 36.454 1.00 36.21 A ATOM 1164 CB ASP A 226 7.769 −4.909 37.8691.00 37.64 A ATOM 1165 CG ASP A 226 6.467 −4.129 37.994 1.00 38.79 AATOM 1166 OD1 ASP A 226 5.726 −4.376 38.967 1.00 40.27 A ATOM 1167 OD2ASP A 226 6.188 −3.265 37.137 1.00 38.30 A ATOM 1168 C ASP A 226 7.301−5.206 35.418 1.00 35.98 A ATOM 1169 O ASP A 226 6.510 −6.133 35.6031.00 36.56 A ATOM 1170 N SER A 227 7.318 −4.437 34.324 1.00 32.04 A ATOM1171 CA SER A 227 6.410 −4.535 33.181 1.00 32.39 A ATOM 1172 CB SER A227 6.159 −6.007 32.791 1.00 33.69 A ATOM 1173 OG SER A 227 5.115 −6.57233.557 1.00 35.74 A ATOM 1174 C SER A 227 5.066 −3.832 33.402 1.00 29.43A ATOM 1175 O SER A 227 4.309 −3.629 32.450 1.00 33.38 A ATOM 1176 N ASNA 228 4.772 −3.453 34.641 1.00 28.29 A ATOM 1177 CA ASN A 228 3.503−2.793 34.955 1.00 29.31 A ATOM 1178 CB ASN A 228 3.029 −3.265 36.3241.00 31.61 A ATOM 1179 CG ASN A 228 2.704 −4.764 36.340 1.00 35.15 AATOM 1180 OD1 ASN A 228 3.127 −5.498 37.241 1.00 36.20 A ATOM 1181 ND2ASN A 228 1.950 −5.211 35.344 1.00 30.27 A ATOM 1182 C ASN A 228 3.588−1.253 34.920 1.00 31.26 A ATOM 1183 O ASN A 228 2.601 −0.565 35.1701.00 31.12 A ATOM 1184 N ALA A 229 4.773 −0.749 34.601 1.00 33.22 A ATOM1185 CA ALA A 229 5.059 0.690 34.505 1.00 33.19 A ATOM 1186 CB ALA A 2294.513 1.428 35.741 1.00 34.56 A ATOM 1187 C ALA A 229 6.575 0.761 34.4781.00 34.91 A ATOM 1188 O ALA A 229 7.230 −0.270 34.609 1.00 33.58 A ATOM1189 N PHE A 230 7.149 1.954 34.313 1.00 32.04 A ATOM 1190 CA PHE A 2308.596 2.068 34.327 1.00 33.83 A ATOM 1191 CB PHE A 230 9.186 1.76432.936 1.00 35.52 A ATOM 1192 CG PHE A 230 8.957 2.842 31.905 1.00 33.95A ATOM 1193 CD1 PHE A 230 9.880 3.882 31.752 1.00 35.30 A ATOM 1194 CD2PHE A 230 7.863 2.781 31.046 1.00 34.06 A ATOM 1195 CE1 PHE A 230 9.7144.835 30.750 1.00 35.01 A ATOM 1196 CE2 PHE A 230 7.683 3.729 30.0381.00 38.15 A ATOM 1197 CZ PHE A 230 8.620 4.767 29.887 1.00 34.58 A ATOM1198 C PHE A 230 9.013 3.459 34.805 1.00 35.37 A ATOM 1199 O PHE A 2308.218 4.392 34.766 1.00 36.39 A ATOM 1200 N SER A 231 10.236 3.57535.301 1.00 36.27 A ATOM 1201 CA SER A 231 10.728 4.870 35.765 1.0037.81 A ATOM 1202 CB SER A 231 10.650 4.976 37.295 1.00 37.18 A ATOM1203 OG SER A 231 11.683 4.254 37.931 1.00 43.77 A ATOM 1204 C SER A 23112.154 5.089 35.298 1.00 39.01 A ATOM 1205 O SER A 231 12.888 4.13934.996 1.00 35.80 A ATOM 1206 N VAL A 232 12.543 6.356 35.219 1.00 37.11A ATOM 1207 CA VAL A 232 13.887 6.694 34.813 1.00 38.94 A ATOM 1208 CBVAL A 232 13.939 7.228 33.362 1.00 40.57 A ATOM 1209 CG1 VAL A 23213.119 8.494 33.233 1.00 44.75 A ATOM 1210 CG2 VAL A 232 15.382 7.48132.964 1.00 42.03 A ATOM 1211 C VAL A 232 14.385 7.755 35.777 1.00 38.34A ATOM 1212 O VAL A 232 13.663 8.696 36.094 1.00 36.03 A ATOM 1213 N ILEA 233 15.598 7.557 36.270 1.00 38.95 A ATOM 1214 CA ILE A 233 16.2138.478 37.216 1.00 41.07 A ATOM 1215 CB ILE A 233 16.231 7.891 38.6501.00 41.10 A ATOM 1216 CG2 ILE A 233 14.824 7.535 39.093 1.00 44.79 AATOM 1217 CG1 ILE A 233 17.123 6.641 38.694 1.00 43.25 A ATOM 1218 CD1ILE A 233 17.451 6.156 40.100 1.00 44.38 A ATOM 1219 C ILE A 233 17.6588.702 36.808 1.00 40.36 A ATOM 1220 O ILE A 233 18.263 7.838 36.181 1.0037.92 A ATOM 1221 N ALA A 234 18.212 9.866 37.144 1.00 40.64 A ATOM 1222CA ALA A 234 19.614 10.109 36.833 1.00 40.47 A ATOM 1223 CB ALA A 23419.992 11.559 37.151 1.00 39.23 A ATOM 1224 C ALA A 234 20.341 9.15037.774 1.00 39.79 A ATOM 1225 O ALA A 234 19.917 8.975 38.917 1.00 41.32A ATOM 1226 N ASP A 235 21.414 8.520 37.311 1.00 39.90 A ATOM 1227 CAASP A 235 22.153 7.591 38.158 1.00 41.21 A ATOM 1228 CB ASP A 235 23.1216.745 37.322 1.00 39.33 A ATOM 1229 CG ASP A 235 23.640 5.537 38.0751.00 40.56 A ATOM 1230 OD1 ASP A 235 23.753 5.614 39.312 1.00 42.34 AATOM 1231 OD2 ASP A 235 23.947 4.511 37.432 1.00 38.58 A ATOM 1232 C ASPA 235 22.949 8.382 39.202 1.00 43.09 A ATOM 1233 O ASP A 235 23.7759.228 38.855 1.00 41.26 A ATOM 1234 N PRO A 236 22.701 8.117 40.494 1.0044.50 A ATOM 1235 CD PRO A 236 21.702 7.176 41.044 1.00 43.52 A ATOM1236 CA PRO A 236 23.412 8.817 41.569 1.00 45.36 A ATOM 1237 CB PRO A236 22.626 8.411 42.818 1.00 45.58 A ATOM 1238 CG PRO A 236 22.159 7.01642.473 1.00 44.95 A ATOM 1239 C PRO A 236 24.888 8.431 41.653 1.00 47.00A ATOM 1240 O PRO A 236 25.691 9.154 42.248 1.00 47.95 A ATOM 1241 N ARGA 237 25.248 7.299 41.051 1.00 46.03 A ATOM 1242 CA ARG A 237 26.6316.835 41.069 1.00 47.36 A ATOM 1243 CB ARG A 237 26.692 5.332 40.7551.00 46.32 A ATOM 1244 CG ARG A 237 26.028 4.434 41.799 1.00 47.54 AATOM 1245 CD ARG A 237 25.908 2.994 41.305 1.00 47.06 A ATOM 1246 NE ARGA 237 25.047 2.902 40.128 1.00 46.65 A ATOM 1247 CZ ARG A 237 24.7051.763 39.529 1.00 47.42 A ATOM 1248 NH1 ARG A 237 25.152 0.598 39.9951.00 45.44 A ATOM 1249 NH2 ARG A 237 23.911 1.792 38.464 1.00 43.05 AATOM 1250 C ARG A 237 27.495 7.597 40.060 1.00 47.83 A ATOM 1251 O ARG A237 28.704 7.383 39.984 1.00 48.93 A ATOM 1252 N GLY A 238 26.873 8.48339.290 1.00 48.46 A ATOM 1253 CA GLY A 238 27.610 9.231 38.288 1.0049.54 A ATOM 1254 C GLY A 238 27.913 8.370 37.076 1.00 50.88 A ATOM 1255O GLY A 238 27.231 7.372 36.826 1.00 50.89 A ATOM 1256 N ASN A 23928.941 8.750 36.323 1.00 51.50 A ATOM 1257 CA ASN A 239 29.335 8.01535.121 1.00 52.78 A ATOM 1258 CB ASN A 239 29.999 8.975 34.122 1.0055.72 A ATOM 1259 CG ASN A 239 30.638 8.252 32.951 1.00 59.41 A ATOM1260 OD1 ASN A 239 30.160 7.203 32.518 1.00 60.19 A ATOM 1261 ND2 ASN A239 31.720 8.819 32.422 1.00 61.11 A ATOM 1262 C ASN A 239 30.275 6.85935.444 1.00 51.17 A ATOM 1263 O ASN A 239 31.491 7.035 35.486 1.00 52.67A ATOM 1264 N THR A 240 29.712 5.674 35.659 1.00 47.62 A ATOM 1265 CATHR A 240 30.511 4.504 35.998 1.00 45.60 A ATOM 1266 CB THR A 240 29.8433.690 37.130 1.00 46.23 A ATOM 1267 OG1 THR A 240 28.571 3.197 36.6851.00 43.74 A ATOM 1268 CG2 THR A 240 29.635 4.566 38.360 1.00 44.58 AATOM 1269 C THR A 240 30.754 3.572 34.816 1.00 45.28 A ATOM 1270 O THR A240 31.700 2.780 34.820 1.00 44.39 A ATOM 1271 N LEU A 241 29.896 3.66033.808 1.00 43.37 A ATOM 1272 CA LEU A 241 30.025 2.819 32.629 1.0043.66 A ATOM 1273 CB LEU A 241 28.658 2.663 31.952 1.00 42.59 A ATOM1274 CG LEU A 241 27.577 1.918 32.740 1.00 42.51 A ATOM 1275 CD1 LEU A241 26.249 2.021 32.011 1.00 40.99 A ATOM 1276 CD2 LEU A 241 27.9870.456 32.911 1.00 41.69 A ATOM 1277 C LEU A 241 31.034 3.353 31.611 1.0042.94 A ATOM 1278 O LEU A 241 31.632 2.574 30.867 1.00 43.16 A ATOM 1279N GLY A 242 31.219 4.672 31.590 1.00 42.04 A ATOM 1280 CA GLY A 24232.127 5.288 30.634 1.00 41.74 A ATOM 1281 C GLY A 242 31.287 5.55329.395 1.00 41.22 A ATOM 1282 O GLY A 242 31.096 6.697 28.970 1.00 40.56A ATOM 1283 N ARG A 243 30.775 4.463 28.833 1.00 39.01 A ATOM 1284 CAARG A 243 29.895 4.469 27.674 1.00 37.25 A ATOM 1285 CB ARG A 243 30.6304.830 26.385 1.00 36.87 A ATOM 1286 CG ARG A 243 29.684 4.953 25.1941.00 36.89 A ATOM 1287 CD ARG A 243 30.380 4.784 23.848 1.00 37.66 AATOM 1288 NE ARG A 243 31.083 3.509 23.744 1.00 36.50 A ATOM 1289 CZ ARGA 243 32.393 3.356 23.901 1.00 39.23 A ATOM 1290 NH1 ARG A 243 33.1674.409 24.162 1.00 38.79 A ATOM 1291 NH2 ARG A 243 32.931 2.142 23.8091.00 39.28 A ATOM 1292 C ARG A 243 29.358 3.046 27.543 1.00 37.57 A ATOM1293 O ARG A 243 30.110 2.073 27.652 1.00 36.86 A ATOM 1294 N GLY A 24428.062 2.916 27.309 1.00 37.87 A ATOM 1295 CA GLY A 244 27.518 1.57927.174 1.00 39.10 A ATOM 1296 C GLY A 244 26.229 1.379 27.932 1.00 37.78A ATOM 1297 O GLY A 244 25.591 2.338 28.363 1.00 36.73 A ATOM 1298 N THRA 245 25.866 0.115 28.122 1.00 37.45 A ATOM 1299 CA THR A 245 24.6180.229 28.776 1.00 35.71 A ATOM 1300 CB THR A 245 23.491 0.395 27.7281.00 38.09 A ATOM 1301 OG1 THR A 245 23.310 0.834 27.015 1.00 38.31 AATOM 1302 CG2 THR A 245 22.180 −0.808 28.400 1.00 37.67 A ATOM 1303 CTHR A 245 24.720 −1.547 29.521 1.00 36.13 A ATOM 1304 O THR A 245 25.351−2.488 29.040 1.00 35.73 A ATOM 1305 N THR A 246 24.106 −1.593 30.6981.00 34.43 A ATOM 1306 CA THR A 246 24.053 −2.804 31.492 1.00 35.99 AATOM 1307 CB THR A 246 24.645 −2.630 32.925 1.00 36.89 A ATOM 1308 OG1THR A 246 26.055 −2.369 32.856 1.00 36.25 A ATOM 1309 CG2 THR A 24624.408 −3.901 33.742 1.00 39.20 A ATOM 1310 C THR A 246 22.578 −3.16331.647 1.00 35.24 A ATOM 1311 O THR A 246 21.762 −2.335 32.078 1.0034.48 A ATOM 1312 N ILE A 247 22.233 −4.388 31.265 1.00 34.48 A ATOM1313 CA ILE A 247 20.868 −4.878 31.421 1.00 33.45 A ATOM 1314 CB ILE A247 20.415 −5.765 30.224 1.00 33.63 A ATOM 1315 CG2 ILE A 247 18.964−6.191 30.421 1.00 35.01 A ATOM 1316 CG1 ILE A 247 20.597 −5.030 28.8831.00 32.43 A ATOM 1317 CD1 ILE A 247 19.686 −3.815 28.676 1.00 34.07 AATOM 1318 C ILE A 247 20.973 −5.777 32.652 1.00 34.92 A ATOM 1319 O ILEA 247 21.689 −6.782 32.624 1.00 33.23 A ATOM 1320 N THR A 248 20.316−5.408 33.744 1.00 36.35 A ATOM 1321 CA THR A 248 20.376 −6.245 34.9381.00 36.95 A ATOM 1322 CB THR A 248 20.753 −5.452 36.214 1.00 39.00 AATOM 1323 OG1 THR A 248 19.665 −4.602 36.596 1.00 47.01 A ATOM 1324 CG2THR A 248 21.974 −4.611 35.977 1.00 32.77 A ATOM 1325 C THR A 248 19.006−6.856 35.139 1.00 37.36 A ATOM 1326 O THR A 248 17.991 −6.149 35.1611.00 35.94 A ATOM 1327 N LEU A 249 18.985 −8.178 35.261 1.00 35.99 AATOM 1328 CA LEU A 249 17.739 −8.910 35.457 1.00 36.92 A ATOM 1329 CBLEU A 249 17.634 −10.069 34.455 1.00 35.98 A ATOM 1330 CG LEU A 24917.708 −9.770 32.949 1.00 34.21 A ATOM 1331 CD1 LEU A 249 17.275 −11.01232.175 1.00 37.00 A ATOM 1332 CD2 LEU A 249 16.796 −8.605 32.595 1.0036.30 A ATOM 1333 C LEU A 249 17.647 −9.479 36.866 1.00 35.98 A ATOM1334 O LEU A 249 18.568 −10.154 37.336 1.00 36.96 A ATOM 1335 N VAL A250 16.541 −9.185 37.537 1.00 36.15 A ATOM 1336 CA VAL A 250 16.274−9.708 38.871 1.00 36.88 A ATOM 1337 CB VAL A 250 15.448 −8.701 39.6911.00 37.61 A ATOM 1338 CG1 VAL A 250 15.135 −9.274 41.075 1.00 39.94 AATOM 1339 CG2 VAL A 250 16.228 −7.395 39.820 1.00 36.44 A ATOM 1340 CVAL A 250 15.449 −10.959 38.557 1.00 36.98 A ATOM 1341 O VAL A 25014.244 −10.883 38.351 1.00 35.45 A ATOM 1342 N LEU A 251 16.120 −12.10738.511 1.00 38.82 A ATOM 1343 CA LEU A 251 15.478 −13.366 38.138 1.0039.06 A ATOM 1344 CB LEU A 251 16.537 −14.470 37.987 1.00 40.36 A ATOM1345 CG LEU A 251 17.640 −14.374 36.924 1.00 40.29 A ATOM 1346 CD1 LEU A251 17.025 −14.052 35.554 1.00 40.30 A ATOM 1347 CD2 LEU A 251 18.643−13.310 37.322 1.00 45.74 A ATOM 1348 C LEU A 251 14.338 −13.918 38.9921.00 40.19 A ATOM 1349 O LEU A 251 14.344 −13.823 40.219 1.00 38.35 AATOM 1350 N LYS A 252 13.368 −14.517 38.308 1.00 40.87 A ATOM 1351 CALYS A 252 12.226 −15.139 38.959 1.00 44.52 A ATOM 1352 CB LYS A 25211.185 −15.568 37.922 1.00 43.89 A ATOM 1353 CG LYS A 252 10.500 −14.43537.163 1.00 44.89 A ATOM 1354 CD LYS A 252 9.645 −15.002 36.032 1.0043.46 A ATOM 1355 CE LYS A 252 8.860 −13.935 35.282 1.00 44.72 A ATOM1356 NZ LYS A 252 7.824 −13.287 36.132 1.00 42.45 A ATOM 1357 C LYS A252 12.768 −16.384 39.658 1.00 47.30 A ATOM 1358 O LYS A 252 13.802−16.931 39.264 1.00 45.15 A ATOM 1359 N GLU A 253 12.068 −16.834 40.6901.00 50.33 A ATOM 1360 CA GLU A 253 12.499 −18.015 41.422 1.00 53.47 AATOM 1361 CB GLU A 253 11.426 −18.423 42.428 1.00 56.08 A ATOM 1362 CGGLU A 253 11.834 −19.568 43.327 1.00 59.63 A ATOM 1363 CD GLU A 25310.674 −20.099 44.135 1.00 62.42 A ATOM 1364 OE1 GLU A 253 9.756 −20.70143.531 1.00 63.41 A ATOM 1365 OE2 GLU A 253 10.676 −19.907 45.371 1.0064.55 A ATOM 1366 C GLU A 253 12.780 −19.185 40.477 1.00 53.70 A ATOM1367 O GLU A 253 13.838 −19.813 40.546 1.00 53.56 A ATOM 1368 N GLU A254 11.833 −19.462 39.588 1.00 55.23 A ATOM 1369 CA GLU A 254 11.957−20.568 38.640 1.00 56.74 A ATOM 1370 CB GLU A 254 10.630 −20.774 37.9051.00 59.86 A ATOM 1371 CG GLU A 254 9.445 −21.073 38.811 1.00 64.81 AATOM 1372 CD GLU A 254 8.165 −21.334 38.035 1.00 67.53 A ATOM 1373 OE1GLU A 254 7.114 −21.563 38.676 1.00 69.99 A ATOM 1374 OE2 GLU A 2548.209 21.311 36.783 1.00 69.08 A ATOM 1375 C GLU A 254 13.065 −20.39637.607 1.00 55.58 A ATOM 1376 O GLU A 254 13.296 −21.290 36.791 1.0055.55 A ATOM 1377 N ALA A 255 13.745 −19.254 37.639 1.00 54.30 A ATOM1378 CA ALA A 255 14.814 −18.970 36.682 1.00 51.41 A ATOM 1379 CB ALA A255 14.560 −17.611 36.016 1.00 51.65 A ATOM 1380 C ALA A 255 16.203−18.990 37.311 1.00 50.31 A ATOM 1381 O ALA A 255 17.177 −18.585 36.6821.00 49.04 A ATOM 1382 N SER A 256 16.292 −19.466 38.550 1.00 50.77 AATOM 1383 CA SER A 256 17.564 −19.531 39.266 1.00 51.61 A ATOM 1384 CBSER A 256 17.366 −20.225 40.622 1.00 53.80 A ATOM 1385 OG SER A 25616.683 −21.464 40.485 1.00 56.72 A ATOM 1386 C SER A 256 18.689 20.21838.486 1.00 52.07 A ATOM 1387 O SER A 256 19.867 −19.906 38.679 1.0050.98 A ATOM 1388 N ASP A 257 18.329 21.145 37.602 1.00 52.71 A ATOM1389 CA ASP A 257 19.321 −21.859 36.799 1.00 54.07 A ATOM 1390 CB ASP A257 18.635 −22.869 35.883 1.00 58.47 A ATOM 1391 CG ASP A 257 18.605−24.255 36.476 1.00 62.55 A ATOM 1392 OD1 ASP A 257 19.699 −24.78636.774 1.00 65.44 A ATOM 1393 OD2 ASP A 257 17.498 −24.811 36.645 1.0065.65 A ATOM 1394 C ASP A 257 20.194 −20.944 35.949 1.00 53.58 A ATOM1395 O ASP A 257 21.346 −21.270 35.659 1.00 52.34 A ATOM 1396 N TYR A258 19.650 −19.805 35.537 1.00 51.92 A ATOM 1397 CA TYR A 258 20.422−18.879 34.720 1.00 52.25 A ATOM 1398 CB TYR A 258 19.498 −17.829 34.0951.00 50.79 A ATOM 1399 CG TYR A 258 18.547 −18.422 33.084 1.00 50.49 AATOM 1400 CD1 TYR A 258 17.204 −18.629 33.394 1.00 49.41 A ATOM 1401 CE1TYR A 258 16.344 −19.230 32.485 1.00 50.81 A ATOM 1402 CD2 TYR A 25819.005 −18.828 31.828 1.00 51.47 A ATOM 1403 CE2 TYR A 258 18.153−19.430 30.912 1.00 51.69 A ATOM 1404 CZ TYR A 258 16.827 −19.629 31.2461.00 52.00 A ATOM 1405 OH TYR A 258 15.988 −20.238 30.344 1.00 52.08 AATOM 1406 C TYR A 258 21.547 −18.219 35.515 1.00 51.93 A ATOM 1407 O TYRA 258 22.375 −17.505 34.957 1.00 53.70 A ATOM 1408 N LEU A 259 21.578−18.476 36.817 1.00 52.47 A ATOM 1409 CA LEU A 259 22.612 −17.927 37.6881.00 52.53 A ATOM 1410 CB LEU A 259 22.080 −17.774 39.115 1.00 52.35 AATOM 1411 CG LEU A 259 20.974 −16.743 39.338 1.00 54.26 A ATOM 1412 CD1LEU A 259 20.439 −16.870 40.757 1.00 53.62 A ATOM 1413 CD2 LEU A 25921.522 −15.341 39.088 1.00 52.70 A ATOM 1414 C LEU A 259 23.834 −18.84337.703 1.00 52.92 A ATOM 1415 O LEU A 259 24.902 −18.459 38.181 1.0052.22 A ATOM 1416 N GLU A 260 23.661 −20.054 37.181 1.00 53.68 A ATOM1417 CA GLU A 260 24.739 −21.039 37.133 1.00 55.47 A ATOM 1418 CB GLU A260 24.158 22.450 36.975 1.00 56.20 A ATOM 1419 CG GLU A 260 23.290−22.910 38.133 1.00 57.13 A ATOM 1420 CD GLU A 260 24.044 −22.947 39.4531.00 59.37 A ATOM 1421 OE1 GLU A 260 25.098 −23.615 39.521 1.00 59.76 AATOM 1422 OE2 GLU A 260 23.580 −22.311 40.426 1.00 59.26 A ATOM 1423 CGLU A 260 25.696 −20.746 35.978 1.00 56.29 A ATOM 1424 O GLU A 26025.265 −20.451 34.861 1.00 55.70 A ATOM 1425 N LEU A 261 26.994 −20.83836.254 1.00 57.25 A ATOM 1426 CA LEU A 261 28.018 −20.571 35.252 1.0058.21 A ATOM 1427 CB LEU A 261 29.407 −20.595 35.896 1.00 58.81 A ATOM1428 CG LEU A 261 29.791 −19.375 36.737 1.00 59.50 A ATOM 1429 CD1 LEU A261 31.140 −19.604 37.404 1.00 59.71 A ATOM 1430 CD2 LEU A 261 29.833−18.145 35.846 1.00 60.31 A ATOM 1431 C LEU A 261 28.004 −21.508 34.0521.00 58.27 A ATOM 1432 O LEU A 261 28.117 −21.057 32.915 1.00 59.29 AATOM 1433 N ASP A 262 27.875 −22.808 34.289 1.00 58.80 A ATOM 1434 CAASP A 262 27.868 −23.751 33.175 1.00 59.21 A ATOM 1435 CB ASP A 26227.896 −25.193 33.692 1.00 61.69 A ATOM 1436 CG ASP A 262 26.714 −25.51734.573 1.00 64.11 A ATOM 1437 OD1 ASP A 262 26.513 24.812 35.585 1.0065.99 A ATOM 1438 OD2 ASP A 262 25.987 −26.482 34.255 1.00 67.06 A ATOM1439 C ASP A 262 26.650 −23.525 32.286 1.00 58.16 A ATOM 1440 O ASP A262 26.699 −23.748 31.078 1.00 57.22 A ATOM 1441 N THR A 263 25.559−23.065 32.886 1.00 57.75 A ATOM 1442 CA THR A 263 24.342 22.804 32.1301.00 57.17 A ATOM 1443 CB THR A 263 23.146 −22.570 33.069 1.00 58.24 AATOM 1444 OG1 THR A 263 22.972 −23.715 33.917 1.00 59.79 A ATOM 1445 CG2THR A 263 21.874 −22.342 32.259 1.00 58.17 A ATOM 1446 C THR A 26324.514 −21.567 31.248 1.00 56.27 A ATOM 1447 O THR A 263 24.284 −21.61830.040 1.00 54.38 A ATOM 1448 N ILE A 264 24.932 −20.464 31.864 1.0055.73 A ATOM 1449 CA ILE A 264 25.125 −19.208 31.148 1.00 56.53 A ATOM1450 CB ILE A 264 25.454 −18.053 32.138 1.00 57.51 A ATOM 1451 CG2 ILE A264 26.717 −18.368 32.915 1.00 57.95 A ATOM 1452 CG1 ILE A 264 25.593−16.728 31.383 1.00 58.53 A ATOM 1453 CD1 ILE A 264 24.274 −16.17330.871 1.00 61.05 A ATOM 1454 C ILE A 264 26.213 −19.296 30.075 1.0056.05 A ATOM 1455 O ILE A 264 26.019 −18.831 28.955 1.00 54.57 A ATOM1456 N LYS A 265 27.352 −19.899 30.406 1.00 57.12 A ATOM 1457 CA LYS A265 28.435 −20.020 29.436 1.00 57.54 A ATOM 1458 CB LYS A 265 29.651−20.711 30.060 1.00 58.26 A ATOM 1459 CG LYS A 265 30.401 −19.831 31.0511.00 59.29 A ATOM 1460 CD LYS A 265 31.778 −20.388 31.414 1.00 60.73 AATOM 1461 CE LYS A 265 31.702 −21.629 32.294 1.00 62.73 A ATOM 1462 NZLYS A 265 31.164 −22.829 31.592 1.00 63.52 A ATOM 1463 C LYS A 26528.011 −20.758 28.172 1.00 57.48 A ATOM 1464 O LYS A 265 28.331 −20.32727.064 1.00 58.37 A ATOM 1465 N ASN A 266 27.283 −21.859 28.335 1.0056.86 A ATOM 1466 CA ASN A 266 26.824 −22.637 27.189 1.00 56.53 A ATOM1467 CB ASN A 266 26.093 −23.899 27.661 1.00 59.04 A ATOM 1468 CG ASN A266 26.207 −25.050 26.671 1.00 62.82 A ATOM 1469 OD1 ASN A 266 27.238−25.728 26.601 1.00 64.55 A ATOM 1470 ND2 ASN A 266 25.151 −25.27125.894 1.00 63.03 A ATOM 1471 C ASN A 266 25.885 −21.793 26.325 1.0055.35 A ATOM 1472 O ASN A 266 25.987 −21.794 25.099 1.00 55.31 A ATOM1473 N LEU A 267 24.974 −21.064 26.967 1.00 53.28 A ATOM 1474 CA LEU A267 24.024 −20.226 26.241 1.00 51.71 A ATOM 1475 CB LEU A 267 22.939−19.702 27.186 1.00 51.22 A ATOM 1476 CG LEU A 267 22.005 −20.747 27.8011.00 50.93 A ATOM 1477 CD1 LEU A 267 21.016 −20.060 28.732 1.00 50.55 AATOM 1478 CD2 LEU A 267 21.274 −21.502 26.701 1.00 49.71 A ATOM 1479 CLEU A 267 24.712 −19.055 25.553 1.00 50.17 A ATOM 1480 O LEU A 26724.414 −18.744 24.402 1.00 49.76 A ATOM 1481 N VAL A 268 25.627 −18.40526.264 1.00 49.61 A ATOM 1482 CA VAL A 268 26.357 −17.278 25.700 1.0050.51 A ATOM 1483 CB VAL A 268 27.350 −16.676 26.719 1.00 50.05 A ATOM1484 CG1 VAL A 268 28.276 −15.695 26.021 1.00 48.90 A ATOM 1485 CG2 VALA 268 26.588 −15.970 27.839 1.00 49.12 A ATOM 1486 C VAL A 268 27.139−17.736 24.475 1.00 52.34 A ATOM 1487 O VAL A 268 27.222 −17.023 23.4771.00 51.12 A ATOM 1488 N LYS A 269 27.715 −18.932 24.559 1.00 54.85 AATOM 1489 CA LYS A 269 28.485 −19.475 23.452 1.00 56.05 A ATOM 1490 CBLYS A 269 29.190 −20.768 23.884 1.00 58.31 A ATOM 1491 CG LYS A 26930.220 −21.273 22.884 1.00 60.99 A ATOM 1492 CD LYS A 269 31.043 −22.43323.445 1.00 62.07 A ATOM 1493 CE LYS A 269 32.103 −22.893 22.445 1.0063.31 A ATOM 1494 NZ LYS A 269 32.919 −24.033 22.962 1.00 63.67 A ATOM1495 C LYS A 269 27.573 −19.741 22.258 1.00 56.18 A ATOM 1496 O LYS A269 27.891 −19.366 21.131 1.00 57.66 A ATOM 1497 N ALA A 270 26.429−20.368 22.508 1.00 55.65 A ATOM 1498 CA ALA A 270 25.489 −20.690 21.4391.00 56.06 A ATOM 1499 CB ALA A 270 24.380 −21.587 21.978 1.00 55.79 AATOM 1500 C ALA A 270 24.873 −19.472 20.761 1.00 56.08 A ATOM 1501 O ALAA 270 24.352 −19.574 19.649 1.00 56.07 A ATOM 1502 N TYR A 271 24.939−18.318 21.416 1.00 56.18 A ATOM 1503 CA TYR A 271 24.343 −17.108 20.8641.00 56.34 A ATOM 1504 CB TYR A 271 23.297 −16.571 21.845 1.00 56.37 AATOM 1505 CG TYR A 271 22.141 −17.506 22.145 1.00 55.02 A ATOM 1506 CD1TYR A 271 21.598 −17.567 23.429 1.00 54.14 A ATOM 1507 CE1 TYR A 27120.500 −18.362 23.708 1.00 55.32 A ATOM 1508 CD2 TYR A 271 21.547−18.278 21.140 1.00 55.39 A ATOM 1509 CE2 TYR A 271 20.438 −19.08021.412 1.00 54.95 A ATOM 1510 CZ TYR A 271 19.920 −19.113 22.700 1.0055.50 A ATOM 1511 OH TYR A 271 18.809 −19.878 22.991 1.00 56.77 A ATOM1512 C TYR A 271 25.324 −15.981 20.524 1.00 57.27 A ATOM 1513 O TYR A271 24.896 −14.873 20.206 1.00 56.68 A ATOM 1514 N SER A 272 26.627−16.247 20.572 1.00 57.63 A ATOM 1515 CA SER A 272 27.601 −15.194 20.2861.00 59.42 A ATOM 1516 CB SER A 272 28.397 −14.873 21.557 1.00 59.53 AATOM 1517 OG SER A 272 29.120 −16.006 22.011 1.00 60.07 A ATOM 1518 CSER A 272 28.575 −15.471 19.138 1.00 60.05 A ATOM 1519 O SER A 27229.495 −14.684 18.897 1.00 59.38 A ATOM 1520 N ALA A 273 28.363 −16.57018.423 1.00 61.51 A ATOM 1521 CA ALA A 273 29.244 −16.943 17.319 1.0062.88 A ATOM 1522 CB ALA A 273 28.935 −18.375 16.875 1.00 63.23 A ATOM1523 C ALA A 273 29.200 −15.997 16.117 1.00 63.61 A ATOM 1524 O ALA A273 30.034 −16.101 15.219 1.00 63.89 A ATOM 1525 N PHE A 274 28.238−15.078 16.093 1.00 65.13 A ATOM 1526 CA PHE A 274 28.127 −14.130 14.9861.00 66.18 A ATOM 1527 CB PHE A 274 26.722 −14.166 14.384 1.00 68.29 AATOM 1528 CG PHE A 274 26.462 −15.365 13.518 1.00 70.72 A ATOM 1529 CD1PHE A 274 26.325 −16.633 14.078 1.00 71.78 A ATOM 1530 CD2 PHE A 27426.345 −15.221 12.136 1.00 72.16 A ATOM 1531 CE1 PHE A 274 26.071−17.748 13.274 1.00 72.69 A ATOM 1532 CE2 PHE A 274 26.092 −16.32411.317 1.00 73.51 A ATOM 1533 CZ PHE A 274 25.953 −17.594 11.888 1.0073.64 A ATOM 1534 C PHE A 274 28.467 −12.688 15.359 1.00 66.07 A ATOM1535 O PHE A 274 28.710 −11.858 14.482 1.00 67.24 A ATOM 1536 N ALA A275 28.479 −12.385 16.652 1.00 65.00 A ATOM 1537 CA ALA A 275 28.792−11.032 17.100 1.00 64.39 A ATOM 1538 CB ALA A 275 28.647 −10.935 18.6191.00 63.46 A ATOM 1539 C ALA A 275 30.212 −10.672 16.681 1.00 63.05 AATOM 1540 O ALA A 275 31.141 −11.443 16.903 1.00 63.35 A ATOM 1541 N ASNA 276 30.374 −9.500 16.076 1.00 62.19 A ATOM 1542 CA ASN A 276 31.687−9.050 15.623 1.00 60.41 A ATOM 1543 CB ASN A 276 31.538 −8.114 14.4221.00 63.49 A ATOM 1544 CG ASN A 276 31.060 −8.837 13.178 1.00 65.72 AATOM 1545 OD1 ASN A 276 31.730 −9.739 12.677 1.00 68.63 A ATOM 1546 ND2ASN A 276 29.896 −8.443 12.671 1.00 66.67 A ATOM 1547 C ASN A 276 32.478−8.357 16.725 1.00 58.37 A ATOM 1548 O ASN A 276 33.331 −7.506 16.4561.00 57.93 A ATOM 1549 N PHE A 277 32.190 −8.737 17.965 1.00 54.60 AATOM 1550 CA PHE A 277 32.862 −8.179 19.131 1.00 51.97 A ATOM 1551 CBPHE A 277 31.989 −7.088 19.753 1.00 51.91 A ATOM 1552 CG PHE A 27731.656 −5.967 18.808 1.00 51.47 A ATOM 1553 CD1 PHE A 277 32.551 −4.91618.611 1.00 52.08 A ATOM 1554 CD2 PHE A 277 30.458 −5.976 18.099 1.0051.35 A ATOM 1555 CE1 PHE A 277 32.256 −3.885 17.718 1.00 51.57 A ATOM1556 CE2 PHE A 277 30.149 −4.955 17.203 1.00 52.12 A ATOM 1557 CZ PHE A277 31.051 −3.905 17.010 1.00 51.46 A ATOM 1558 C PHE A 277 33.071−9.315 20.131 1.00 50.36 A ATOM 1559 O PHE A 277 32.320 −10.286 20.1391.00 46.88 A ATOM 1560 N PRO A 278 34.100 −9.216 20.980 1.00 50.28 AATOM 1561 CD PRO A 278 35.153 −8.187 21.072 1.00 50.22 A ATOM 1562 CAPRO A 278 34.325 −10.291 21.954 1.00 50.16 A ATOM 1563 CB PRO A 27835.724 −9.989 22.470 1.00 49.80 A ATOM 1564 CG PRO A 278 35.768 −8.47922.431 1.00 50.62 A ATOM 1565 C PRO A 278 33.283 −10.298 23.079 1.0050.37 A ATOM 1566 O PRO A 278 32.877 −9.236 23.565 1.00 50.51 A ATOM1567 N ILE A 279 32.839 −11.489 23.476 1.00 50.25 A ATOM 1568 CA ILE A279 31.871 −11.609 24.567 1.00 51.04 A ATOM 1569 CB ILE A 279 30.549−12.285 24.125 1.00 51.79 A ATOM 1570 CG2 ILE A 279 29.604 −12.40525.320 1.00 52.28 A ATOM 1571 CG1 ILE A 279 29.859 −11.457 23.037 1.0053.44 A ATOM 1572 CD1 ILE A 279 30.363 −11.729 21.632 1.00 56.53 A ATOM1573 C ILE A 279 32.484 −12.433 25.694 1.00 50.74 A ATOM 1574 O ILE A279 32.937 −13.561 25.481 1.00 51.89 A ATOM 1575 N TYR A 280 32.482−11.864 26.894 1.00 49.59 A ATOM 1576 CA TYR A 280 33.058 −12.504 28.0701.00 50.16 A ATOM 1577 CB TYR A 280 34.070 −11.557 28.717 1.00 50.09 AATOM 1578 CG TYR A 280 35.165 −11.095 27.792 1.00 51.60 A ATOM 1579 CD1TYR A 280 36.252 −11.920 27.501 1.00 51.31 A ATOM 1580 CE1 TYR A 28037.264 −11.500 26.647 1.00 52.44 A ATOM 1581 CD2 TYR A 280 35.116 −9.83227.201 1.00 51.35 A ATOM 1582 CE2 TYR A 280 36.122 −9.402 26.344 1.0052.57 A ATOM 1583 CZ TYR A 280 37.193 −10.242 26.073 1.00 52.17 A ATOM1584 OH TYR A 280 38.190 −9.828 25.223 1.00 53.86 A ATOM 1585 C TYR A280 32.024 −12.885 29.124 1.00 50.79 A ATOM 1586 O TYR A 280 30.915−12.353 29.144 1.00 49.68 A ATOM 1587 N VAL A 281 32.416 −13.799 30.0091.00 50.64 A ATOM 1588 CA VAL A 281 31.571 −14.244 31.111 1.00 50.82 AATOM 1589 CB VAL A 281 30.900 −15.607 30.808 1.00 50.44 A ATOM 1590 CG1VAL A 281 29.885 −15.459 29.691 1.00 50.15 A ATOM 1591 CG2 VAL A 28131.951 −16.629 30.428 1.00 52.25 A ATOM 1592 C VAL A 281 32.449 −14.37632.359 1.00 51.99 A ATOM 1593 O VAL A 281 33.454 −15.088 32.346 1.0052.81 A ATOM 1594 N TRP A 282 32.081 −13.669 33.424 1.00 52.59 A ATOM1595 CA TRP A 282 32.832 −13.705 34.677 1.00 54.91 A ATOM 1596 CB TRP A282 32.166 −12.781 35.696 1.00 54.67 A ATOM 1597 CG TRP A 282 33.000−12.444 36.899 1.00 55.63 A ATOM 1598 CD2 TRP A 282 34.314 −11.86736.905 1.00 55.87 A ATOM 1599 CE2 TRP A 282 34.670 −11.650 38.253 1.0056.26 A ATOM 1600 CE3 TRP A 282 35.223 −11.509 35.900 1.00 56.93 A ATOM1601 CD1 TRP A 282 32.628 −12.556 38.210 1.00 55.67 A ATOM 1602 NE1 TRPA 282 33.624 −12.079 39.028 1.00 55.84 A ATOM 1603 CZ2 TRP A 282 35.901−11.089 38.624 1.00 56.57 A ATOM 1604 CZ3 TRP A 282 36.448 −10.95036.269 1.00 56.51 A ATOM 1605 CH2 TRP A 282 36.772 −10.746 37.619 1.0056.85 A ATOM 1606 C TRP A 282 32.798 −15.149 35.171 1.00 56.74 A ATOM1607 O TRP A 282 31.789 −15.598 35.708 1.00 56.36 A ATOM 1608 N SER A283 33.901 −15.870 34.983 1.00 59.27 A ATOM 1609 CA SER A 283 33.979−17.276 35.375 1.00 61.20 A ATOM 1610 CB SER A 283 34.242 −18.133 34.1371.00 61.51 A ATOM 1611 OG SER A 283 33.307 −17.840 33.113 1.00 64.73 AATOM 1612 C SER A 283 35.039 −17.588 36.429 1.00 62.33 A ATOM 1613 O SERA 283 35.887 −16.756 36.749 1.00 62.30 A ATOM 1614 N SER A 284 34.983−18.809 36.955 1.00 64.03 A ATOM 1615 CA SER A 284 35.923 −19.257 37.9761.00 65.62 A ATOM 1616 CB SER A 284 35.234 −19.292 39.341 1.00 65.36 AATOM 1617 OG SER A 284 34.134 −20.184 39.324 1.00 65.82 A ATOM 1618 CSER A 284 36.469 −20.643 37.640 1.00 66.68 A ATOM 1619 O SER A 28435.886 −21.375 36.835 1.00 66.59 A ATOM 1620 N LYS A 285 37.587 −21.00438.264 1.00 67.92 A ATOM 1621 CA LYS A 285 38.208 −22.301 38.016 1.0069.26 A ATOM 1622 CB LYS A 285 39.173 −22.186 36.831 1.00 70.51 A ATOM1623 CG LYS A 285 39.780 −23.502 36.359 1.00 72.05 A ATOM 1624 CD LYS A285 40.592 −23.286 35.084 1.00 73.12 A ATOM 1625 CE LYS A 285 41.195−24.584 34.563 1.00 74.70 A ATOM 1626 NZ LYS A 285 41.976 −24.375 33.3071.00 74.12 A ATOM 1627 C LYS A 285 38.950 −22.810 39.249 1.00 69.18 AATOM 1628 O LYS A 285 38.934 −22.084 40.265 1.00 69.24 A ATOM 1629 CBVAL A 330 41.691 −18.076 39.440 1.00 65.70 A ATOM 1630 CG1 VAL A 33042.175 −16.694 38.995 1.00 65.89 A ATOM 1631 CG2 VAL A 330 42.767−18.794 40.241 1.00 66.10 A ATOM 1632 C VAL A 330 39.284 −17.407 39.4001.00 65.01 A ATOM 1633 O VAL A 330 38.717 −18.144 38.596 1.00 65.36 AATOM 1634 N VAL A 330 40.012 −19.256 40.874 1.00 65.49 A ATOM 1635 CAVAL A 330 40.408 −17.941 40.289 1.00 65.73 A ATOM 1636 N TRP A 33138.973 −16.123 39.553 1.00 65.05 A ATOM 1637 CA TRP A 331 37.914 −15.47138.782 1.00 65.10 A ATOM 1638 CB TRP A 331 36.972 −14.715 39.727 1.0067.00 A ATOM 1639 CG TRP A 331 35.754 −15.482 40.159 1.00 69.49 A ATOM1640 CD2 TRP A 331 35.509 −16.054 41.450 1.00 70.82 A ATOM 1641 CE2 TRPA 331 34.228 −16.648 41.409 1.00 71.28 A ATOM 1642 CE3 TRP A 331 36.246−16.120 42.640 1.00 71.31 A ATOM 1643 CD1 TRP A 331 34.644 −15.75039.406 1.00 70.76 A ATOM 1644 NE1 TRP A 331 33.723 −16.448 40.151 1.0071.62 A ATOM 1645 CZ2 TRP A 331 33.668 −17.302 42.516 1.00 71.95 A ATOM1646 CZ3 TRP A 331 35.689 −16.771 43.742 1.00 72.37 A ATOM 1647 CH2 TRPA 331 34.413 −17.351 43.670 1.00 72.81 A ATOM 1648 C TRP A 331 38.466−14.493 37.742 1.00 64.34 A ATOM 1649 O TRP A 331 39.305 −13.647 38.0581.00 62.86 A ATOM 1650 N ASP A 332 37.983 −14.609 36.505 1.00 63.43 AATOM 1651 CA ASP A 332 38.419 −13.724 35.425 1.00 63.19 A ATOM 1652 CBASP A 332 39.826 −14.117 34.955 1.00 64.46 A ATOM 1653 CG ASP A 33240.680 −12.913 34.585 1.00 66.04 A ATOM 1654 OD1 ASP A 332 40.245−12.092 33.748 1.00 66.86 A ATOM 1655 OD2 ASP A 332 41.795 −12.78935.134 1.00 67.88 A ATOM 1656 C ASP A 332 37.450 −13.784 34.238 1.0061.65 A ATOM 1657 O ASP A 332 36.635 −14.703 34.133 1.00 60.08 A ATOM1658 N TRP A 333 37.536 −12.793 33.353 1.00 59.80 A ATOM 1659 CA TRP A333 36.686 −12.759 32.169 1.00 58.58 A ATOM 1660 CB TRP A 333 36.805−11.410 31.452 1.00 56.51 A ATOM 1661 CG TRP A 333 36.236 −10.257 32.2161.00 54.90 A ATOM 1662 CD2 TRP A 333 34.864 −10.056 32.574 1.00 53.44 AATOM 1663 CE2 TRP A 333 34.788 −8.833 33.276 1.00 54.00 A ATOM 1664 CE3TRP A 333 33.690 −10.792 32.370 1.00 53.96 A ATOM 1665 CD1 TRP A 33336.920 −9.179 32.704 1.00 54.32 A ATOM 1666 NE1 TRP A 333 36.057 −8.31933.341 1.00 53.95 A ATOM 1667 CZ2 TRP A 333 33.577 −8.326 33.776 1.0052.51 A ATOM 1668 CZ3 TRP A 333 32.485 −10.286 32.868 1.00 53.62 A ATOM1669 CH2 TRP A 333 32.442 −9.067 33.561 1.00 52.56 A ATOM 1670 C TRP A333 37.119 −13.873 31.221 1.00 58.90 A ATOM 1671 O TRP A 333 38.286−13.952 30.832 1.00 58.73 A ATOM 1672 N GLU A 334 36.178 −14.738 30.8561.00 58.96 A ATOM 1673 CA GLU A 334 36.473 −15.839 29.951 1.00 59.53 AATOM 1674 CB GLU A 334 35.928 −17.149 30.516 1.00 60.54 A ATOM 1675 CGGLU A 334 36.514 −18.382 29.859 1.00 62.48 A ATOM 1676 CD GLU A 33435.800 −19.652 30.265 1.00 64.73 A ATOM 1677 OE1 GLU A 334 35.527−19.825 31.470 1.00 65.42 A ATOM 1678 OE2 GLU A 334 35.515 −20.48129.376 1.00 65.95 A ATOM 1679 C GLU A 334 35.847 −15.581 28.584 1.0059.18 A ATOM 1680 O GLU A 334 34.631 −15.440 28.466 1.00 58.69 A ATOM1681 N LEU A 335 36.679 −15.523 27.552 1.00 58.58 A ATOM 1682 CA LEU A335 36.190 −15.282 26.200 1.00 59.22 A ATOM 1683 CB LEU A 335 37.372−15.091 25.248 1.00 59.10 A ATOM 1684 CG LEU A 335 37.052 −14.785 23.7861.00 59.02 A ATOM 1685 CD1 LEU A 335 36.417 −13.406 23.679 1.00 57.75 AATOM 1686 CD2 LEU A 335 38.336 −14.850 22.968 1.00 59.37 A ATOM 1687 CLEU A 335 35.321 −16.440 25.713 1.00 59.48 A ATOM 1688 O LEU A 33535.745 −17.596 25.727 1.00 59.46 A ATOM 1689 N MET A 336 34.102 −16.12225.290 1.00 59.17 A ATOM 1690 CA MET A 336 33.169 −17.123 24.787 1.0060.97 A ATOM 1691 CB MET A 336 31.767 −16.862 25.349 1.00 61.75 A ATOM1692 CG MET A 336 31.675 −16.920 26.870 1.00 62.57 A ATOM 1693 SD MET A336 32.033 −18.560 27.531 1.00 64.96 A ATOM 1694 CE MET A 336 33.730−18.383 27.925 1.00 64.61 A ATOM 1695 C MET A 336 33.152 −17.018 23.2641.00 61.64 A ATOM 1696 O MET A 336 32.490 −17.796 22.578 1.00 61.35 AATOM 1697 N ASN A 337 33.906 −16.037 22.772 1.00 62.88 A ATOM 1698 CAASN A 337 34.059 −15.710 21.355 1.00 64.29 A ATOM 1699 CB ASN A 33733.962 −16.952 20.467 1.00 66.06 A ATOM 1700 CG ASN A 337 34.470 −16.68919.063 1.00 67.89 A ATOM 1701 OD1 ASN A 337 35.553 −16.125 18.888 1.0069.70 A ATOM 1702 ND2 ASN A 337 33.699 −17.096 18.056 1.00 68.64 A ATOM1703 C ASN A 337 33.031 −14.675 20.909 1.00 63.60 A ATOM 1704 O ASN A337 32.161 −15.008 20.078 1.00 63.24 A ATOM 1705 OXT ASN A 337 33.111−13.536 21.409 1.00 63.10 A ATOM 1706 OH2 WAT W 1 24.847 0.476 24.7711.00 32.16 W ATOM 1707 OH2 WAT W 2 14.818 −5.124 21.757 1.00 33.62 WATOM 1708 OH2 WAT W 3 8.952 −0.499 21.618 1.00 36.76 W ATOM 1709 OH2 WATW 4 30.911 −29.736 33.235 1.00 79.70 W ATOM 1710 OH2 WAT W 5 2.130−7.313 33.720 1.00 38.37 W ATOM 1711 OH2 WAT W 6 28.011 −7.152 33.2871.00 42.07 W ATOM 1712 OH2 WAT W 7 8.579 −18.872 35.549 1.00 63.18 WATOM 1713 OH2 WAT W 8 2.341 0.845 32.049 1.00 34.97 W ATOM 1714 OH2 WATW 9 21.534 −5.717 25.523 1.00 37.67 W ATOM 1715 OH2 WAT W 10 18.884−3.010 18.470 1.00 47.48 W ATOM 1716 OH2 WAT W 11 23.026 8.416 28.8201.00 46.39 W ATOM 1717 OH2 WAT W 12 21.101 5.291 21.278 1.00 45.73 WATOM 1718 OH2 WAT W 13 24.002 −5.027 27.071 1.00 36.22 W ATOM 1719 OH2WAT W 14 3.071 7.977 31.329 1.00 40.54 W ATOM 1720 OH2 WAT W 15 27.4417.441 22.808 1.00 33.79 W ATOM 1721 OH2 WAT W 16 2.583 7.339 37.179 1.0065.71 W ATOM 1722 OH2 WAT W 17 18.873 −6.771 17.897 1.00 44.39 W ATOM1723 OH2 WAT W 18 2.483 −1.807 31.770 1.00 32.18 W ATOM 1724 OH2 WAT W19 7.866 −10.916 34.603 1.00 41.35 W ATOM 1725 OH2 WAT W 20 32.781 7.38324.625 1.00 44.35 W ATOM 1726 OH2 WAT W 21 7.711 −20.828 22.884 1.0041.13 W ATOM 1727 OH2 WAT W 22 21.547 12.590 16.946 1.00 70.66 W ATOM1728 OH2 WAT W 23 18.791 −5.014 25.314 1.00 30.54 W ATOM 1729 C5* NEC N1 14.630 −1.760 21.594 1.00 42.82 N ATOM 1730 O5* NEC N 1 15.095 −2.73722.487 1.00 39.14 N ATOM 1731 N5* NEC N 1 13.379 −1.892 20.912 1.0047.41 N ATOM 1732 C51 NEC N 1 12.204 −2.356 21.609 1.00 47.38 N ATOM1733 C52 NEC N 1 11.143 −2.661 20.558 1.00 47.12 N ATOM 1734 C4* NEC N 115.422 −0.530 21.323 1.00 40.52 N ATOM 1735 O4* NEC N 1 15.959 0.04322.481 1.00 38.34 N ATOM 1736 C3* NEC N 1 16.575 −0.698 20.373 1.0040.65 N ATOM 1737 O3* NEC N 1 16.181 −0.610 19.032 1.00 40.85 N ATOM1738 C2* NEC N 1 17.553 0.382 20.858 1.00 41.27 N ATOM 1739 O2* NEC N 117.221 1.611 20.223 1.00 41.96 N ATOM 1740 C1* NEC N 1 17.345 0.39022.405 1.00 37.25 N ATOM 1741 N9 NEC N 1 18.267 −0.595 23.039 1.00 37.54N ATOM 1742 C8 NEC N 1 17.971 −1.829 23.616 1.00 35.13 N ATOM 1743 N7NEC N 1 19.036 −2.426 24.091 1.00 34.96 N ATOM 1744 C5 NEC N 1 20.093−1.540 23.825 1.00 35.95 N ATOM 1745 C6 NEC N 1 21.496 −1.571 24.0981.00 35.57 N ATOM 1746 N6 NEC N 1 22.092 −2.599 24.726 1.00 36.19 N ATOM1747 N1 NEC N 1 22.270 −0.493 23.697 1.00 37.88 N ATOM 1748 C2 NEC N 121.683 0.546 23.065 1.00 34.37 N ATOM 1749 N3 NEC N 1 20.394 0.69322.754 1.00 34.33 N ATOM 1750 C4 NEC N 1 19.632 −0.399 23.165 1.00 36.84N

TABLE 2 ATOMIC STRUCTURE COORDINATE DATA OBTAINED FROM X-RAY DIFFRACTIONFROM THE LIGAND BINDNG POCKET OF FORM 1 OF THE LIGAND BINDING DOMAIN OFGRP94 (RESIDUES 48-316 OF SEQ ID NO: 6) IN COMPLEX WITH NECA ATOM 106 NMET A 85 2.451 −5.703 22.762 1.00 36.19 A ATOM 107 CA MET A 85 3.6526.287 22.182 1.00 36.75 A ATOM 108 CB MET A 85 4.609 −5.184 21.693 1.0038.82 A ATOM 109 CG MET A 85 5.974 −5.703 21.210 1.00 41.23 A ATOM 110SD MET A 85 6.976 −4.519 20.233 1.00 43.17 A ATOM 111 CE MET A 85 7.035−3.128 21.369 1.00 45.81 A ATOM 112 C MET A 85 4.374 −7.186 23.177 1.0036.50 A ATOM 113 O MET A 85 4.917 −8.229 22.797 1.00 37.23 A ATOM 258 NGLU A 103 18.584 −12.439 22.390 1.00 35.80 A ATOM 259 CA GLU A 10318.172 −11.084 22.033 1.00 36.77 A ATOM 260 CB GLU A 103 16.667 −10.92722.244 1.00 38.94 A ATOM 261 CG GLU A 103 15.845 −11.797 21.293 1.0044.14 A ATOM 262 CD GLU A 103 16.277 −11.613 19.843 1.00 48.10 A ATOM263 OE1 GLU A 103 16.200 −10.471 19.344 1.00 47.73 A ATOM 264 OE2 GLU A103 16.706 −12.603 19.205 1.00 48.75 A ATOM 265 C GLU A 103 18.933−9.987 22.764 1.00 37.18 A ATOM 266 O GLU A 103 19.260 −8.956 22.1671.00 38.35 A ATOM 267 N LEU A 104 19.213 −10.181 24.052 1.00 36.53 AATOM 268 CA LEU A 104 19.967 −9.168 24.782 1.00 34.96 A ATOM 269 CB LEUA 104 20.051 −9.514 26.266 1.00 36.16 A ATOM 270 CG LEU A 104 18.724−9.575 27.021 1.00 34.61 A ATOM 271 CD1 LEU A 104 18.994 −9.853 28.4901.00 36.29 A ATOM 272 CD2 LEU A 104 17.971 −8.254 26.855 1.00 37.01 AATOM 273 C LEU A 104 21.369 −9.081 24.183 1.00 36.79 A ATOM 274 O LEU A104 21.956 7.995 24.098 1.00 35.24 A ATOM 289 N ASN A 107 21.038 −7.27920.880 1.00 35.94 A ATOM 290 CA ASN A 107 20.841 −5.846 21.110 1.0038.70 A ATOM 291 CB ASN A 107 19.829 −5.623 22.241 1.00 37.51 A ATOM 292CG ASN A 107 18.406 −5.947 21.826 1.00 36.75 A ATOM 293 OD1 ASN A 10717.509 −6.028 22.667 1.00 38.88 A ATOM 294 ND2 ASN A 107 18.188 −6.12020.531 1.00 32.53 A ATOM 295 C ASN A 107 22.168 −5.161 21.457 1.00 38.73A ATOM 296 O ASN A 107 22.425 −4.029 21.030 1.00 38.05 A ATOM 297 N ALAA 108 23.010 −5.854 22.224 1.00 37.25 A ATOM 298 CA ALA A 108 24.306−5.319 22.616 1.00 37.11 A ATOM 299 CB ALA A 108 24.944 −6.218 23.6891.00 35.32 A ATOM 300 C ALA A 108 25.208 −5.220 21.384 1.00 38.35 A ATOM301 O ALA A 108 25.949 −4.248 21.219 1.00 36.53 A ATOM 308 N ASP A 11024.249 −4.776 18.322 1.00 37.55 A ATOM 309 CA ASP A 110 23.765 −3.64517.523 1.00 38.97 A ATOM 310 CB ASP A 110 22.236 −3.578 17.545 1.0039.76 A ATOM 311 CG ASP A 110 21.580 −4.651 16.686 1.00 42.87 A ATOM 312OD1 ASP A 110 20.344 −4.769 16.743 1.00 41.85 A ATOM 313 OD2 ASP A 11022.290 −5.373 15.956 1.00 46.31 A ATOM 314 C ASP A 110 24.327 −2.32618.060 1.00 36.57 A ATOM 315 O ASP A 110 24.750 −1.469 17.284 1.00 37.95A ATOM 316 N ALA A 111 24.315 −2.173 19.385 1.00 36.03 A ATOM 317 CA ALAA 111 24.827 −0.969 20.047 1.00 35.99 A ATOM 318 CB ALA A 111 24.577−1.047 21.560 1.00 34.35 A ATOM 319 C ALA A 111 26.315 −0.808 19.7791.00 36.64 A ATOM 320 O ALA A 111 26.801 0.310 19.586 1.00 34.89 A ATOM579 N VAL A 147 24.143 −8.039 31.844 1.00 35.57 A ATOM 580 CA VAL A 14724.662 −8.102 30.478 1.00 36.33 A ATOM 581 CB VAL A 147 23.599 −8.55329.478 1.00 35.23 A ATOM 582 CG1 VAL A 147 24.188 −8.592 28.082 1.0037.63 A ATOM 583 CG2 VAL A 147 23.096 −9.941 29.858 1.00 37.09 A ATOM584 C VAL A 147 25.087 −6.669 30.160 1.00 36.34 A ATOM 585 O VAL A 14724.250 −5.778 29.980 1.00 34.90 A ATOM 593 N ASP A 149 27.425 −3.93827.840 1.00 37.49 A ATOM 594 CA ASP A 149 28.112 −3.694 26.593 1.0037.69 A ATOM 595 CB ASP A 149 27.166 −3.865 25.388 1.00 38.06 A ATOM 596CG ASP A 149 26.100 −2.779 25.299 1.00 39.30 A ATOM 597 OD1 ASP A 14926.449 −1.599 25.056 1.00 39.14 A ATOM 598 OD2 ASP A 149 24.905 −3.10725.471 1.00 37.40 A ATOM 599 C ASP A 149 28.687 −2.286 26.637 1.00 37.48A ATOM 600 O ASP A 149 28.236 −1.442 27.412 1.00 37.39 A ATOM 612 N VALA 152 26.921 2.139 21.990 1.00 38.14 A ATOM 613 CA VAL A 152 26.5393.547 21.916 1.00 36.85 A ATOM 614 CB VAL A 152 25.663 3.800 20.660 1.0036.97 A ATOM 615 CG1 VAL A 152 24.359 3.030 20.784 1.00 34.47 A ATOM 616CG2 VAL A 152 25.398 5.312 20.473 1.00 36.38 A ATOM 617 C VAL A 15225.797 4.071 23.150 1.00 38.24 A ATOM 618 O VAL A 152 25.862 5.26423.457 1.00 37.41 A ATOM 619 N GLY A 153 25.090 3.188 23.854 1.00 35.89A ATOM 620 CA GLY A 153 24.344 3.613 25.023 1.00 33.35 A ATOM 621 C GLYA 153 23.074 4.371 24.641 1.00 33.66 A ATOM 622 O GLY A 153 22.769 4.51923.458 1.00 33.60 A ATOM 623 N MET A 154 22.339 4.844 25.645 1.00 33.73A ATOM 624 CA MET A 154 21.098 5.583 25.435 1.00 35.02 A ATOM 625 CB META 154 19.881 4.687 25.713 1.00 35.45 A ATOM 626 CG MET A 154 19.6723.579 24.711 1.00 36.62 A ATOM 627 SD MET A 154 18.279 2.497 25.176 1.0039.95 A ATOM 628 CE MET A 154 19.165 1.378 26.258 1.00 38.04 A ATOM 629C MET A 154 20.991 6.802 26.353 1.00 34.74 A ATOM 630 O MET A 154 21.3306.734 27.532 1.00 34.79 A ATOM 654 N GLU A 158 16.061 8.597 25.142 1.0035.85 A ATOM 655 CA GLU A 158 16.003 7.314 24.438 1.00 38.21 A ATOM 656CB GLU A 158 17.372 6.982 23.852 1.00 37.42 A ATOM 657 CG GLU A 15817.828 8.013 22.815 1.00 42.18 A ATOM 658 CD GLU A 158 19.127 7.64922.141 1.00 41.21 A ATOM 659 OE1 GLU A 158 20.131 7.435 22.843 1.0042.08 A ATOM 660 OE2 GLU A 158 19.153 7.587 20.897 1.00 47.22 A ATOM 661C GLU A 158 15.511 6.174 25.346 1.00 37.73 A ATOM 662 O GLU A 158 14.7925.283 24.895 1.00 38.81 A ATOM 678 N ALA A 161 11.730 6.786 25.413 1.0034.61 A ATOM 679 CA ALA A 161 11.091 6.516 24.128 1.00 35.52 A ATOM 680CB ALA A 161 11.512 7.573 23.110 1.00 35.95 A ATOM 681 C ALA A 16111.304 5.129 23.523 1.00 34.49 A ATOM 682 O ALA A 161 10.349 4.47623.115 1.00 36.23 A ATOM 683 N ASN A 162 12.555 4.698 23.457 1.00 34.89A ATOM 684 CA ASN A 162 12.898 3.422 22.839 1.00 36.90 A ATOM 685 CB ASNA 162 14.414 3.331 22.691 1.00 37.47 A ATOM 686 CG ASN A 162 14.9614.415 21.785 1.00 42.03 A ATOM 687 OD1 ASN A 162 14.383 5.505 21.6881.00 43.34 A ATOM 688 ND2 ASN A 162 16.075 4.134 21.125 1.00 40.97 AATOM 689 C ASN A 162 12.354 2.196 23.551 1.00 37.70 A ATOM 690 O ASN A162 12.039 1.186 22.908 1.00 37.59 A ATOM 691 N LEU A 163 12.232 2.27724.869 1.00 37.09 A ATOM 692 CA LEU A 163 11.722 1.135 25.624 1.00 38.10A ATOM 693 CB LEU A 163 12.587 0.906 26.864 1.00 35.47 A ATOM 694 CG LEUA 163 14.093 0.725 26.645 1.00 34.53 A ATOM 695 CD1 LEU A 163 14.7920.602 28.003 1.00 34.16 A ATOM 696 CD2 LEU A 163 14.361 −0.506 25.7701.00 37.46 A ATOM 697 C LEU A 163 10.262 1.297 26.035 1.00 38.71 A ATOM698 O LEU A 163 9.541 0.309 26.209 1.00 38.36 A ATOM 718 N ALA A 1678.904 3.353 19.167 1.00 53.09 A ATOM 719 CA ALA A 167 9.809 4.375 18.6581.00 55.59 A ATOM 720 CB ALA A 167 11.139 4.277 19.400 1.00 55.40 A ATOM721 C ALA A 167 10.062 4.404 17.155 1.00 57.83 A ATOM 722 O ALA A 16710.185 5.483 16.575 1.00 57.75 A ATOM 723 N LYS A 168 10.148 3.23716.522 1.00 61.19 A ATOM 724 CA LYS A 168 10.418 3.186 15.085 1.00 63.46A ATOM 725 CB LYS A 168 11.717 2.421 14.825 1.00 64.87 A ATOM 726 CG LYSA 168 12.978 3.188 15.198 1.00 67.57 A ATOM 727 CD LYS A 168 13.1034.488 14.403 1.00 68.54 A ATOM 728 CE LYS A 168 13.123 4.234 12.898 1.0069.62 A ATOM 729 NZ LYS A 168 14.290 3.409 12.479 1.00 70.51 A ATOM 730C LYS A 168 9.318 2.600 14.213 1.00 64.19 A ATOM 731 O LYS A 168 8.2762.159 14.701 1.00 64.53 A ATOM 742 N THR A 171 9.191 −1.575 14.421 1.0059.30 A ATOM 743 CA THR A 171 8.412 −2.158 15.505 1.00 59.38 A ATOM 744CB THR A 171 8.724 −1.453 16.850 1.00 58.69 A ATOM 745 OG1 THR A 1717.737 −1.810 17.824 1.00 60.23 A ATOM 746 CG2 THR A 171 8.750 0.04116.675 1.00 61.04 A ATOM 747 C THR A 171 6.911 −2.127 15.221 1.00 58.48A ATOM 748 O THR A 171 6.161 −2.973 15.707 1.00 58.04 A ATOM 924 N GLY A196 14.281 −6.880 15.898 1.00 43.45 A ATOM 925 CA GLY A 196 14.991−6.496 17.105 1.00 43.03 A ATOM 926 C GLY A 196 14.139 −6.021 18.2691.00 41.78 A ATOM 927 O GLY A 196 14.488 −5.043 18.935 1.00 39.12 A ATOM928 N VAL A 197 13.032 −6.717 18.529 1.00 39.22 A ATOM 929 CA VAL A 19712.139 −6.353 19.629 1.00 37.80 A ATOM 930 CB VAL A 197 10.659 −6.28219.155 1.00 37.78 A ATOM 931 CG1 VAL A 197 10.498 −5.196 18.101 1.0039.35 A ATOM 932 CG2 VAL A 197 10.212 −7.633 18.592 1.00 39.35 A ATOM933 C VAL A 197 12.228 −7.321 20.821 1.00 36.08 A ATOM 934 O VAL A 19711.379 −7.292 21.708 1.00 33.97 A ATOM 935 N GLY A 198 13.264 −8.15920.838 1.00 35.72 A ATOM 936 CA GLY A 198 13.445 −9.133 21.910 1.0034.04 A ATOM 937 C GLY A 198 13.722 −8.639 23.324 1.00 35.00 A ATOM 938O GLY A 198 13.688 −9.427 24.269 1.00 32.54 A ATOM 939 N PHE A 19914.009 −7.349 23.500 1.00 34.83 A ATOM 940 CA PHE A 199 14.267 −6.84124.841 1.00 33.95 A ATOM 941 CB PHE A 199 14.400 −5.308 24.804 1.0035.31 A ATOM 942 CG PHE A 199 14.375 −4.663 26.159 1.00 35.09 A ATOM 943CD1 PHE A 199 15.521 −4.614 26.946 1.00 34.35 A ATOM 944 CD2 PHE A 19913.183 −4.130 26.660 1.00 35.32 A ATOM 945 CE1 PHE A 199 15.490 −4.04428.225 1.00 34.68 A ATOM 946 CE2 PHE A 199 13.131 −3.561 27.929 1.0034.37 A ATOM 947 CZ PHE A 199 14.295 −3.518 28.720 1.00 35.36 A ATOM 948C PHE A 199 13.165 −7.250 25.829 1.00 33.31 A ATOM 949 O PHE A 19913.449 −7.652 26.961 1.00 31.85 A ATOM 950 N TYR A 200 11.913 −7.17825.395 1.00 33.78 A ATOM 951 CA TYR A 200 10.799 −7.515 26.270 1.0035.65 A ATOM 952 CB TYR A 200 9.479 −7.090 25.621 1.00 35.72 A ATOM 953CG TYR A 200 9.432 −5.610 25.293 1.00 36.99 A ATOM 954 CD1 TYR A 2009.404 −4.645 26.304 1.00 35.32 A ATOM 955 CE1 TYR A 200 9.413 −3.26725.990 1.00 37.74 A ATOM 956 CD2 TYR A 200 9.465 −5.177 23.971 1.0038.74 A ATOM 957 CE2 TYR A 200 9.474 −3.826 23.654 1.00 40.33 A ATOM 958CZ TYR A 200 9.449 −2.877 24.665 1.00 38.60 A ATOM 959 OH TYR A 2009.463 −1.542 24.326 1.00 43.22 A ATOM 960 C TYR A 200 10.716 −8.98226.707 1.00 35.83 A ATOM 961 O TYR A 200 11.0067 −9.284 27.712 1.0032.96 A ATOM 1036 N VAL A 211 17.875 −1.148 33.430 1.00 34.49 A ATOM1037 CA VAL A 211 18.791 0.793 32.366 1.00 32.75 A ATOM 1038 CB VAL A211 18.038 −0.519 31.036 1.00 33.29 A ATOM 1039 CG1 VAL A 211 19.032−0.186 29.942 1.00 32.50 A ATOM 1040 CG2 VAL A 211 17.211 −1.759 30.6081.00 32.87 A ATOM 1041 C VAL A 211 19.558 0.456 32.786 1.00 34.30 A ATOM1042 O VAL A 211 18.958 1.504 33.015 1.00 31.53 A ATOM 1133 N TRP A 22316.848 2.945 34.029 1.00 34.41 A ATOM 1134 CA TRP A 223 15.462 2.67933.643 1.00 33.72 A ATOM 1135 CB TRP A 223 15.391 2.404 32.130 1.0034.35 A ATOM 1136 CG TRP A 223 14.032 2.052 31.545 1.00 34.80 A ATOM1137 CD2 TRP A 223 13.444 0.743 31.460 1.00 33.67 A ATOM 1138 CE2 TRP A223 12.243 0.868 30.726 1.00 34.03 A ATOM 1139 CE3 TRP A 223 13.825−0.526 31.926 1.00 35.44 A ATOM 1140 CD1 TRP A 223 13.173 2.897 30.8901.00 33.40 A ATOM 1141 NE1 TRP A 223 12.096 2.190 30.393 1.00 34.12 AATOM 1142 CZ2 TRP A 223 11.414 −0.234 30.448 1.00 33.49 A ATOM 1143 CZ3TRP A 223 12.992 −1.627 31.645 1.00 32.64 A ATOM 1144 CH2 TRP A 22311.813 −1.468 30.914 1.00 31.20 A ATOM 1145 C TRP A 223 15.150 1.40634.438 1.00 34.21 A ATOM 1146 O TRP A 223 16.015 0.537 34.559 1.00 34.33A ATOM 1298 N THR A 245 25.866 0.115 28.122 1.00 37.45 A ATOM 1299 CATHR A 245 24.618 −0.229 28.776 1.00 35.71 A ATOM 1300 CB THR A 24523.491 −0.395 27.728 1.00 38.09 A ATOM 1301 OG1 THR A 245 23.310 0.83427.015 1.00 38.31 A ATOM 1302 CG2 THR A 245 22.180 −0.808 28.400 1.0037.67 A ATOM 1303 C THR A 245 24.720 −1.547 29.521 1.00 36.13 A ATOM1304 O THR A 245 25.351 2.488 29.040 1.00 35.73 A ATOM 1312 N ILE A 24722.233 −4.388 31.265 1.00 34.48 A ATOM 1313 CA ILE A 247 20.868 4.87831.421 1.00 33.45 A ATOM 1314 CB ILE A 247 20.415 −5.765 30.224 1.0033.63 A ATOM 1315 CG2 ILE A 247 18.964 −6.191 30.421 1.00 35.01 A ATOM1316 CG1 ILE A 247 20.597 −5.030 28.883 1.00 32.43 A ATOM 1317 CD1 ILE A247 19.686 −3.815 28.676 1.00 34.07 A ATOM 1318 C ILE A 247 20.973−5.777 32.652 1.00 34.92 A ATOM 1319 O ILE A 247 21.689 −6.782 32.6241.00 33.23 A ATOM 1706 OH2 WAT W 1 24.847 0.476 24.771 1.00 32.16 W ATOM1718 OH2 WAT W 13 24.002 −5.027 27.071 1.00 36.22 W ATOM 1722 OH2 WAT W17 18.873 −6.771 17.897 1.00 44.39 W ATOM 1707 OH2 WAT W 2 14.818 −5.12421.757 1.00 33.62 W ATOM 1708 OH2 WAT W 3 8.952 −0.499 21.618 1.00 36.76W ATOM 1714 OH2 WAT W 9 21.534 −5.717 25.523 1.00 37.67 W ATOM 1715 OH2WAT W 10 18.884 −3.010 18.470 1.00 47.48 W ATOM 1717 OH2 WAT W 12 21.1015.291 21.278 1.00 45.73 W ATOM 1728 OH2 WAT W 23 18.791 −5.014 25.3141.00 30.54 W ATOM 1729 C5* NEC N 1 14.630 −1.760 21.594 1.00 42.82 NATOM 1730 O5* NEC N 1 15.095 −2.737 22.487 1.00 39.14 N ATOM 1731 N5*NEC N 1 13.379 −1.892 20.912 1.00 47.41 N ATOM 1732 C51 NEC N 1 12.204−2.356 21.609 1.00 47.38 N ATOM 1733 C52 NEC N 1 11.143 −2.661 20.5581.00 47.12 N ATOM 1734 C4* NEC N 1 15.422 −0.530 21.323 1.00 40.52 NATOM 1735 O4* NEC N 1 15.959 0.043 22.481 1.00 38.34 N ATOM 1736 C3* NECN 1 16.575 −0.698 20.373 1.00 40.65 N ATOM 1737 O3* NEC N 1 16.181−0.610 19.032 1.00 40.85 N ATOM 1738 C2* NEC N 1 17.553 0.382 20.8581.00 41.27 N ATOM 1739 O2* NEC N 1 17.221 1.611 20.223 1.00 41.96 N ATOM1740 C1* NEC N 1 17.345 0.390 22.405 1.00 37.25 N ATOM 1741 N9 NEC N 118.267 −0.595 23.039 1.00 37.54 N ATOM 1742 C8 NEC N 1 17.971 −1.82923.616 1.00 35.13 N ATOM 1743 N7 NEC N 1 19.036 −2.426 24.091 1.00 34.96N ATOM 1744 C5 NEC N 1 20.093 −1.540 23.825 1.00 35.95 N ATOM 1745 C6NEC N 1 21.496 −1.571 24.098 1.00 35.57 N ATOM 1746 N6 NEC N 1 22.092−2.599 24.726 1.00 36.19 N ATOM 1747 N1 NEC N 1 22.270 −0.493 23.6971.00 37.88 N ATOM 1748 C2 NEC N 1 21.683 0.546 23.065 1.00 34.37 N ATOM1749 N3 NEC N 1 20.394 0.693 22.754 1.00 34.33 N ATOM 1750 C4 NEC N 119.632 −0.399 23.165 1.00 36.84 N

TABLE 3 ATOMIC STRUCTURE COORDINATE DATA OBTAINED FROM X-RAY DIFFRACTIONFROM FORM 2 OF THE LIGAND BINDING DOMAIN OF GRP94 (RESIDUES 48-316 OFSEQ ID NO: 6) IN COMPLEX WITH NECA ATOM 1 CB LYS A 72 33.507 −16.29672.107 1.00 69.73 A ATOM 2 CG LYS A 72 33.483 −15.288 73.248 1.00 70.11A ATOM 3 CD LYS A 72 33.524 −15.987 74.598 1.00 71.49 A ATOM 4 CE LYS A72 33.552 −14.988 75.745 1.00 72.79 A ATOM 5 NZ LYS A 72 34.793 −14.16575.741 1.00 72.91 A ATOM 6 C LYS A 72 31.931 −15.062 70.609 1.00 67.84 AATOM 7 O LYS A 72 30.984 −15.730 70.200 1.00 68.80 A ATOM 8 N LYS A 7234.390 −14.667 70.454 1.00 69.61 A ATOM 9 CA LYS A 72 33.324 −15.67970.715 1.00 69.35 A ATOM 10 N SER A 73 31.820 −13.789 70.987 1.00 65.64A ATOM 11 CA SER A 73 30.555 −13.056 70.944 1.00 63.60 A ATOM 12 CB SERA 73 30.007 −12.863 72.361 1.00 64.12 A ATOM 13 OG SER A 73 28.825−12.079 72.353 1.00 63.95 A ATOM 14 C SER A 73 30.779 −11.692 70.2931.00 61.79 A ATOM 15 O SER A 73 31.712 −10.975 70.662 1.00 63.42 A ATOM16 N GLU A 74 29.927 −11.331 69.335 1.00 57.06 A ATOM 17 CA GLU A 7430.072 −10.054 68.642 1.00 53.07 A ATOM 18 CB GLU A 74 30.972 −10.24467.417 1.00 53.91 A ATOM 19 CG GLU A 74 31.283 −8.971 66.657 1.00 55.81A ATOM 20 CD GLU A 74 32.438 −9.142 65.689 1.00 57.46 A ATOM 21 OE1 GLUA 74 32.445 −10.135 64.931 1.00 56.10 A ATOM 22 OE2 GLU A 74 33.338−8.276 65.684 1.00 59.85 A ATOM 23 C GLU A 74 28.732 −9.431 68.231 1.0050.14 A ATOM 24 O GLU A 74 27.853 −10.111 67.694 1.00 48.22 A ATOM 25 NALA A 75 28.591 −8.132 68.489 1.00 45.60 A ATOM 26 CA ALA A 75 27.369−7.395 68.175 1.00 43.37 A ATOM 27 CB ALA A 75 27.066 −6.390 69.287 1.0043.20 A ATOM 28 C ALA A 75 27.453 −6.673 66.834 1.00 41.49 A ATOM 29 OALA A 75 28.530 −6.237 66.413 1.00 40.95 A ATOM 30 N PHE A 76 26.311−6.544 66.167 1.00 37.00 A ATOM 31 CA PHE A 76 26.254 −5.881 64.868 1.0036.00 A ATOM 32 CB PHE A 76 26.259 −6.904 63.721 1.00 34.46 A ATOM 33 CGPHE A 76 27.463 −7.804 63.685 1.00 32.78 A ATOM 34 CD1 PHE A 76 27.530−8.932 64.494 1.00 33.06 A ATOM 35 CD2 PHE A 76 28.515 −7.539 62.8101.00 33.42 A ATOM 36 CE1 PHE A 76 28.631 −9.792 64.432 1.00 34.56 A ATOM37 CE2 PHE A 76 29.624 −8.392 62.739 1.00 34.73 A ATOM 38 CZ PHE A 7629.681 −9.519 63.549 1.00 32.36 A ATOM 39 C PHE A 76 24.989 −5.04664.695 1.00 36.16 A ATOM 40 O PHE A 76 23.945 −5.337 65.282 1.00 35.60 AATOM 41 N ALA A 77 25.089 −4.015 63.867 1.00 34.33 A ATOM 42 CA ALA A 7723.936 −3.188 63.553 1.00 33.00 A ATOM 43 CB ALA A 77 24.306 −1.71563.580 1.00 34.28 A ATOM 44 C ALA A 77 23.555 −3.609 62.138 1.00 32.33 AATOM 45 O ALA A 77 24.427 −3.973 61.337 1.00 28.99 A ATOM 46 N PHE A 7822.261 −3.590 61.837 1.00 29.93 A ATOM 47 CA PHE A 78 21.802 −3.94860.503 1.00 28.73 A ATOM 48 CB PHE A 78 20.275 −4.111 60.465 1.00 26.59A ATOM 49 CG PHE A 78 19.768 −5.389 61.083 1.00 24.26 A ATOM 50 CD1 PHEA 78 19.297 −5.409 62.398 1.00 23.07 A ATOM 51 CD2 PHE A 78 19.725−6.564 60.341 1.00 23.49 A ATOM 52 CE1 PHE A 78 18.788 −6.577 62.9571.00 23.95 A ATOM 53 CE2 PHE A 78 19.217 −7.738 60.888 1.00 20.39 A ATOM54 CZ PHE A 78 18.746 −7.746 62.201 1.00 21.62 A ATOM 55 C PHE A 7822.183 −2.843 59.522 1.00 30.30 A ATOM 56 O PHE A 78 22.275 −1.67059.893 1.00 29.90 A ATOM 57 N GLN A 79 22.402 −3.218 58.268 1.00 30.62 AATOM 58 CA GLN A 79 22.717 −2.236 57.240 1.00 31.01 A ATOM 59 CB GLN A79 23.051 −2.941 55.925 1.00 29.52 A ATOM 60 CG GLN A 79 23.557 −2.02654.825 1.00 33.63 A ATOM 61 CD GLN A 79 23.726 −2.756 53.499 1.00 36.38A ATOM 62 OE1 GLN A 79 23.884 −3.975 53.468 1.00 34.09 A ATOM 63 NE2 GLNA 79 23.702 −2.006 52.397 1.00 37.63 A ATOM 64 C GLN A 79 21.436 −1.40757.082 1.00 30.67 A ATOM 65 O GLN A 79 20.330 −1.942 57.201 1.00 28.06 AATOM 66 N ALA A 80 21.582 −0.113 56.816 1.00 29.05 A ATOM 67 CA ALA A 8020.426 0.769 56.664 1.00 30.86 A ATOM 68 CB ALA A 80 20.873 2.122 56.0981.00 29.60 A ATOM 69 C ALA A 80 19.318 0.176 55.785 1.00 32.06 A ATOM 70O ALA A 80 18.152 0.131 56.188 1.00 33.23 A ATOM 71 N GLU A 81 19.685−0.275 54.588 1.00 31.42 A ATOM 72 CA GLU A 81 18.728 −0.849 53.651 1.0031.65 A ATOM 73 CB GLU A 81 19.421 −1.265 52.352 1.00 33.71 A ATOM 74 CGGLU A 81 19.909 −0.117 51.489 1.00 39.87 A ATOM 75 CD GLU A 81 21.1900.516 52.003 1.00 44.19 A ATOM 76 OE1 GLU A 81 21.693 1.447 51.334 1.0046.79 A ATOM 77 OE2 GLU A 81 21.693 0.089 53.069 1.00 46.52 A ATOM 78 CGLU A 81 17.978 −2.054 54.203 1.00 30.64 A ATOM 79 O GLU A 81 16.822−2.287 53.841 1.00 28.44 A ATOM 80 N VAL A 82 18.634 −2.838 55.053 1.0026.89 A ATOM 81 CA VAL A 82 17.971 −4.001 55.615 1.00 27.07 A ATOM 82 CBVAL A 82 18.968 −4.957 56.299 1.00 25.85 A ATOM 83 CG1 VAL A 82 18.209−6.070 57.020 1.00 21.09 A ATOM 84 CG2 VAL A 82 19.919 −5.551 55.2501.00 24.54 A ATOM 85 C VAL A 82 16.910 −3.545 56.614 1.00 27.82 A ATOM86 O VAL A 82 15.856 −4.166 56.729 1.00 26.27 A ATOM 87 N ASN A 8317.188 −2.463 57.337 1.00 25.48 A ATOM 88 CA ASN A 83 16.206 −1.93858.278 1.00 26.67 A ATOM 89 CB ASN A 83 16.738 −0.705 59.019 1.00 24.92A ATOM 90 CG ASN A 83 17.707 −1.056 60.135 1.00 28.46 A ATOM 91 OD1 ASNA 83 17.559 −2.080 60.813 1.00 25.85 A ATOM 92 ND2 ASN A 83 18.695−0.191 60.347 1.00 26.16 A ATOM 93 C ASN A 83 14.968 −1.536 57.471 1.0029.03 A ATOM 94 O ASN A 83 13.835 −1.833 57.861 1.00 28.84 A ATOM 95 NARG A 84 15.196 −0.857 56.348 1.00 29.73 A ATOM 96 CA ARG A 84 14.104−0.413 55.487 1.00 32.14 A ATOM 97 CB ARG A 84 14.631 0.475 54.351 1.0035.80 A ATOM 98 CG ARG A 84 15.480 1.633 54.839 1.00 40.52 A ATOM 99 CDARG A 84 15.714 2.721 53.787 1.00 42.66 A ATOM 100 NE ARG A 84 16.5723.762 54.354 1.00 42.69 A ATOM 101 CZ ARG A 84 17.871 3.890 54.101 1.0042.60 A ATOM 102 NH1 ARG A 84 18.480 3.053 53.267 1.00 39.66 A ATOM 103NH2 ARG A 84 18.573 4.828 54.724 1.00 43.08 A ATOM 104 C ARG A 84 13.389−1.621 54.905 1.00 32.38 A ATOM 105 O ARG A 84 12.159 −1.684 54.902 1.0033.16 A ATOM 106 N MET A 85 14.162 −2.584 54.413 1.00 31.59 A ATOM 107CA MET A 85 13.576 −3.789 53.847 1.00 30.94 A ATOM 108 CB MET A 8514.675 −4.744 53.361 1.00 33.35 A ATOM 109 CG MET A 85 14.176 −6.14152.996 1.00 34.44 A ATOM 110 SD MET A 85 15.307 −7.026 51.900 1.00 36.89A ATOM 111 CE MET A 85 16.747 −7.176 52.979 1.00 35.72 A ATOM 112 C META 85 12.695 −4.482 54.882 1.00 29.99 A ATOM 113 O MET A 85 11.614 −4.96554.559 1.00 30.02 A ATOM 114 N MET A 86 13.156 −4.529 56.126 1.00 28.78A ATOM 115 CA MET A 86 12.375 −5.169 57.174 1.00 29.00 A ATOM 116 CB META 86 13.120 −5.130 58.517 1.00 29.54 A ATOM 117 CG MET A 86 14.276−6.125 58.632 1.00 29.72 A ATOM 118 SD MET A 86 14.755 −6.426 60.3611.00 31.17 A ATOM 119 CE MET A 86 16.134 −5.288 60.552 1.00 32.15 A ATOM120 C MET A 86 11.014 −4.486 57.309 1.00 29.93 A ATOM 121 O MET A 869.984 −5.152 57.369 1.00 29.47 A ATOM 122 N ALA A 87 11.014 −3.15757.339 1.00 30.60 A ATOM 123 CA ALA A 87 9.772 −2.394 57.467 1.00 29.51A ATOM 124 CB ALA A 87 10.092 −0.909 57.636 1.00 30.35 A ATOM 125 C ALAA 87 8.824 −2.600 56.277 1.00 28.78 A ATOM 126 O ALA A 87 7.612 −2.71256.447 1.00 26.77 A ATOM 127 N LEU A 88 9.375 −2.646 55.072 1.00 29.52 AATOM 128 CA LEU A 88 8.548 −2.843 53.888 1.00 31.42 A ATOM 129 CB LEU A88 9.395 −2.740 52.621 1.00 33.48 A ATOM 130 CG LEU A 88 9.893 −1.34252.258 1.00 35.63 A ATOM 131 CD1 LEU A 88 10.754 −1.430 51.005 1.0035.88 A ATOM 132 CD2 LEU A 88 8.696 −0.412 52.040 1.00 36.34 A ATOM 133C LEU A 88 7.869 −4.206 53.936 1.00 31.94 A ATOM 134 O LEU A 88 6.684−4.338 53.637 1.00 30.13 A ATOM 135 N ILE A 89 8.635 −5.222 54.318 1.0031.82 A ATOM 136 CA ILE A 89 8.111 −6.572 54.395 1.00 31.63 A ATOM 137CB ILE A 89 9.241 −7.577 54.669 1.00 32.58 A ATOM 138 CG2 ILE A 89 8.660−8.945 54.995 1.00 32.00 A ATOM 139 CG1 ILE A 89 10.181 −7.628 53.4581.00 32.05 A ATOM 140 CD1 ILE A 89 11.358 −8.573 53.632 1.00 32.05 AATOM 141 C ILE A 89 7.058 −6.690 55.479 1.00 32.50 A ATOM 142 O ILE A 895.972 −7.226 55.250 1.00 32.33 A ATOM 143 N ILE A 90 7.380 −6.183 56.6621.00 32.87 A ATOM 144 CA ILE A 90 6.459 −6.246 57.780 1.00 34.48 A ATOM145 CB ILE A 90 7.081 −5.577 59.028 1.00 34.93 A ATOM 146 CG2 ILE A 906.002 −5.258 60.056 1.00 34.14 A ATOM 147 CG1 ILE A 90 8.173 −6.49359.596 1.00 36.87 A ATOM 148 CD1 ILE A 90 8.941 −5.920 60.780 1.00 37.62A ATOM 149 C ILE A 90 5.128 −5.588 57.432 1.00 36.73 A ATOM 150 O ILE A90 4.072 −6.211 57.541 1.00 3603 A ATOM 151 N ASN A 91 5.184 −4.33656.998 1.00 37.77 A ATOM 152 CA ASN A 91 3.980 −3.601 56.648 1.00 41.16A ATOM 153 CB ASN A 91 4.346 −2.148 56.335 1.00 44.99 A ATOM 154 CG ASNA 91 4.612 −1.334 57.599 1.00 49.01 A ATOM 155 OD1 ASN A 91 3.678 −0.94358.303 1.00 52.43 A ATOM 156 ND2 ASN A 91 5.886 −1.091 57.901 1.00 50.00A ATOM 157 C ASN A 91 3.200 −4.232 55.492 1.00 40.60 A ATOM 158 O ASN A91 1.976 −4.138 55.441 1.00 41.03 A ATOM 159 N SER A 92 3.907 −4.89254.580 1.00 39.25 A ATOM 160 CA SER A 92 3.264 −5.536 53.444 1.00 37.85A ATOM 161 CB SER A 92 4.308 −5.936 52.402 1.00 40.00 A ATOM 162 OG SERA 92 3.763 −6.868 51.480 1.00 44.69 A ATOM 163 C SER A 92 2.451 −6.76953.825 1.00 35.95 A ATOM 164 O SER A 92 1.382 −7.007 53.262 1.00 37.27 AATOM 165 N LEU A 93 2.959 −7.553 54.771 1.00 33.26 A ATOM 166 CA LEU A93 2.291 −8.775 55.212 1.00 32.62 A ATOM 167 CB LEU A 93 3.303 −9.92455.299 1.00 31.56 A ATOM 168 CG LEU A 93 4.171 −10.233 54.074 1.00 33.63A ATOM 169 CD1 LEU A 93 5.176 −11.333 54.432 1.00 32.56 A ATOM 170 CD2LEU A 93 3.296 −10.665 52.901 1.00 32.73 A ATOM 171 C LEU A 93 1.635−8.606 56.580 1.00 34.15 A ATOM 172 O LEU A 93 1.292 −9.593 57.241 1.0033.21 A ATOM 173 N TYR A 94 1.458 −7.365 57.011 1.00 35.00 A ATOM 174 CATYR A 94 0.873 −7.124 58.320 1.00 38.40 A ATOM 175 CB TYR A 94 0.692−5.626 58.565 1.00 40.76 A ATOM 176 CG TYR A 94 0.583 −5.308 60.030 1.0045.83 A ATOM 177 CD1 TYR A 94 1.712 −5.354 60.851 1.00 48.25 A ATOM 178CE1 TYR A 94 1.618 −5.121 62.220 1.00 50.09 A ATOM 179 CD2 TYR A 94−0.649 −5.018 60.615 1.00 47.99 A ATOM 180 CE2 TYR A 94 −0.754 −4.78261.986 1.00 50.05 A ATOM 181 CZ TYR A 94 0.384 −4.837 62.780 1.00 50.62A ATOM 182 OH TYR A 94 0.293 −4.618 64.136 1.00 54.45 A ATOM 183 C TYR A94 −0.462 −7.834 58.552 1.00 38.49 A ATOM 184 O TYR A 94 −0.748 −8.27959.665 1.00 36.56 A ATOM 185 N LYS A 95 −1.277 −7.946 57.507 1.00 38.51A ATOM 186 CA LYS A 95 −2.575 −8.594 57.648 1.00 39.01 A ATOM 187 CB LYSA 95 −3.640 −7.790 56.904 1.00 41.67 A ATOM 188 CG LYS A 95 −3.935−6.434 57.534 1.00 44.54 A ATOM 189 CD LYS A 95 −4.232 −6.572 59.0241.00 46.17 A ATOM 190 CE LYS A 95 −4.967 −5.356 59.573 1.00 48.95 A ATOM191 NZ LYS A 95 −4.210 −4.085 59.382 1.00 50.46 A ATOM 192 C LYS A 95−2.601 −10.047 57.190 1.00 38.34 A ATOM 193 O LYS A 95 −3.671 −10.65457.078 1.00 37.55 A ATOM 194 N ASN A 96 −1.417 −10.596 56.934 1.00 36.93A ATOM 195 CA ASN A 96 −1.266 −11.981 56.499 1.00 36.51 A ATOM 196 CBASN A 96 −1.174 −12.052 54.971 1.00 39.38 A ATOM 197 CG ASN A 96 −2.487−11.701 54.291 1.00 41.91 A ATOM 198 OD1 ASN A 96 −3.414 −12.512 54.2491.00 43.88 A ATOM 199 ND2 ASN A 96 −2.576 −10.485 53.769 1.00 43.40 AATOM 200 C ASN A 96 0.012 −12.523 57.130 1.00 36.61 A ATOM 201 O ASN A96 0.885 −13.057 56.447 1.00 34.41 A ATOM 202 N LYS A 97 0.101 −12.37858.447 1.00 35.10 A ATOM 203 CA LYS A 97 1.261 −12.810 59.209 1.00 34.80A ATOM 204 CB LYS A 97 0.995 −12.605 60.705 1.00 33.16 A ATOM 205 CG LYSA 97 0.770 −11.150 61.086 1.00 34.93 A ATOM 206 CD LYS A 97 0.336−11.005 62.542 1.00 40.66 A ATOM 207 CE LYS A 97 0.437 −9.561 63.0201.00 41.82 A ATOM 208 NZ LYS A 97 −0.331 −8.616 62.171 1.00 43.56 A ATOM209 C LYS A 97 1.685 −14.248 58.955 1.00 35.24 A ATOM 210 O LYS A 972.881 −14.539 58.874 1.00 35.69 A ATOM 211 N GLU A 98 0.712 −15.14458.818 1.00 35.04 A ATOM 212 CA GLU A 98 1.013 −16.553 58.605 1.00 35.34A ATOM 213 CB GLU A 98 −0.281 −17.353 58.378 1.00 37.17 A ATOM 214 CGGLU A 98 −1.069 −17.009 57.120 1.00 42.04 A ATOM 215 CD GLU A 98 −2.062−15.872 57.321 1.00 44.24 A ATOM 216 OE1 GLU A 98 −2.874 −15.632 56.3991.00 47.71 A ATOM 217 OE2 GLU A 98 −2.037 −15.219 58.388 1.00 43.42 AATOM 218 C GLU A 98 2.009 −16.836 57.476 1.00 33.13 A ATOM 219 O GLU A98 2.654 −17.886 57.462 1.00 32.97 A ATOM 220 N ILE A 99 2.144 −15.90356.541 1.00 31.36 A ATOM 221 CA ILE A 99 3.070 −16.083 55.428 1.00 32.00A ATOM 222 CB ILE A 99 2.947 −14.914 54.411 1.00 33.50 A ATOM 223 CG2ILE A 99 4.033 −15.002 53.345 1.00 34.71 A ATOM 224 CG1 ILE A 99 1.571−14.975 53.739 1.00 38.54 A ATOM 225 CD1 ILE A 99 1.392 −13.998 52.5901.00 40.32 A ATOM 226 C ILE A 99 4.514 −16.213 55.923 1.00 31.10 A ATOM227 O ILE A 99 5.409 −16.604 55.168 1.00 29.83 A ATOM 228 N PHE A 1004.743 −15.899 57.197 1.00 29.56 A ATOM 229 CA PHE A 100 6.085 −16.01157.746 1.00 28.31 A ATOM 230 CB PHE A 100 6.141 −15.512 59.204 1.0026.69 A ATOM 231 CG PHE A 100 5.755 −16.553 60.228 1.00 27.65 A ATOM 232CD1 PHE A 100 4.439 −16.670 60.664 1.00 27.08 A ATOM 233 CD2 PHE A 1006.715 −17.430 60.744 1.00 28.13 A ATOM 234 CE1 PHE A 100 4.074 −17.65061.605 1.00 28.84 A ATOM 235 CE2 PHE A 100 6.367 −18.413 61.681 1.0028.66 A ATOM 236 CZ PHE A 100 5.042 −18.522 62.112 1.00 29.66 A ATOM 237C PHE A 100 6.519 −17.476 57.684 1.00 27.86 A ATOM 238 O PHE A 100 7.671−17.782 57.375 1.00 26.69 A ATOM 239 N LEU A 101 5.586 −18.378 57.9671.00 26.61 A ATOM 240 CA LEU A 101 5.894 −19.803 57.966 1.00 27.71 AATOM 241 CB LEU A 101 4.761 −20.594 58.630 1.00 28.29 A ATOM 242 CG LEUA 101 5.048 −22.074 58.906 1.00 31.62 A ATOM 243 CD1 LEU A 101 6.310−22.197 59.757 1.00 31.66 A ATOM 244 CD2 LEU A 101 3.863 −22.711 59.6221.00 32.58 A ATOM 245 C LEU A 101 6.141 −20.324 56.556 1.00 27.21 A ATOM246 O LEU A 101 6.978 −21.204 56.354 1.00 29.92 A ATOM 247 N ARG A 1025.414 −19.782 55.583 1.00 26.05 A ATOM 248 CA ARG A 102 5.587 −20.18554.192 1.00 27.16 A ATOM 249 CB ARG A 102 4.637 −19.414 53.274 1.0027.20 A ATOM 250 CG ARG A 102 4.666 −19.895 51.824 1.00 28.59 A ATOM 251CD ARG A 102 3.882 −18.953 50.906 1.00 29.28 A ATOM 252 NE ARG A 1024.616 −17.720 50.638 1.00 32.58 A ATOM 253 CZ ARG A 102 4.129 −16.68649.956 1.00 36.08 A ATOM 254 NH1 ARG A 102 2.893 −16.728 49.473 1.0033.94 A ATOM 255 NH2 ARG A 102 4.888 −15.618 49.734 1.00 33.41 A ATOM256 C ARG A 102 7.014 −19.886 53.755 1.00 26.97 A ATOM 257 O ARG A 1027.674 −20.724 53.139 1.00 27.51 A ATOM 258 N GLU A 103 7.478 −18.68054.077 1.00 26.40 A ATOM 259 CA GLU A 103 8.820 −18.243 53.715 1.0025.90 A ATOM 260 CB GLU A 103 8.967 −16.740 53.973 1.00 27.03 A ATOM 261CG GLU A 103 8.084 −15.892 53.074 1.00 30.54 A ATOM 262 CD GLU A 1038.227 −16.277 51.610 1.00 34.94 A ATOM 263 OE1 GLU A 103 9.367 −16.27151.096 1.00 36.36 A ATOM 264 OE2 GLU A 103 7.200 −16.592 50.971 1.0036.06 A ATOM 265 C GLU A 103 9.933 −19.006 54.424 1.00 27.65 A ATOM 266O GLU A 103 10.949 −19.329 53.811 1.00 28.59 A ATOM 267 N LEU A 1049.762 −19.289 55.714 1.00 27.46 A ATOM 268 CA LEU A 104 10.789 −20.02756.437 1.00 28.00 A ATOM 269 CB LEU A 104 10.446 −20.109 57.929 1.0028.75 A ATOM 270 CG LEU A 104 10.344 −18.774 58.677 1.00 27.18 A ATOM271 CD1 LEU A 104 10.066 −19.029 60.150 1.00 29.40 A ATOM 272 CD2 LEU A104 11.640 −17.990 58.510 1.00 26.56 A ATOM 273 C LEU A 104 10.920−21.433 55.846 1.00 28.30 A ATOM 274 O LEU A 104 12.021 −21.973 55.7391.00 26.95 A ATOM 275 N ILE A 105 9.792 −22.023 55.466 1.00 27.22 A ATOM276 CA ILE A 105 9.805 −23.355 54.874 1.00 29.99 A ATOM 277 CB ILE A 1058.365 −23.937 54.770 1.00 31.43 A ATOM 278 CG2 ILE A 105 8.328 −25.11953.793 1.00 29.81 A ATOM 279 CG1 ILE A 105 7.901 −24.372 56.167 1.0031.88 A ATOM 280 CD1 ILE A 105 6.493 −24.890 56.231 1.00 34.12 A ATOM281 C ILE A 105 10.458 −23.277 53.499 1.00 30.30 A ATOM 282 O ILE A 10511.252 −24.138 53.125 1.00 30.17 A ATOM 283 N SER A 106 10.138 −22.22052.762 1.00 29.33 A ATOM 284 CA SER A 106 10.710 −22.007 51.438 1.0030.77 A ATOM 285 CB SER A 106 10.097 −20.754 50.810 1.00 31.84 A ATOM286 OG SER A 106 10.662 −20.496 49.542 1.00 39.34 A ATOM 287 C SER A 10612.237 −21.857 51.545 1.00 30.70 A ATOM 288 O SER A 106 12.981 −22.45850.767 1.00 29.18 A ATOM 289 N ASN A 107 12.700 −21.060 52.509 1.0028.35 A ATOM 290 CA ASN A 107 14.139 −20.866 52.705 1.00 28.64 A ATOM291 CB ASN A 107 14.409 −19.875 53.846 1.00 27.62 A ATOM 292 CG ASN A107 14.083 −18.434 53.472 1.00 29.03 A ATOM 293 OD1 ASN A 107 14.153−17.529 54.317 1.00 29.37 A ATOM 294 ND2 ASN A 107 13.737 −18.210 52.2111.00 23.85 A ATOM 295 C ASN A 107 14.791 −22.212 53.044 1.00 28.48 AATOM 296 O ASN A 107 15.881 −22.530 52.563 1.00 27.33 A ATOM 297 N ALA A108 14.116 −22.995 53.881 1.00 27.12 A ATOM 298 CA ALA A 108 14.628−24.301 54.278 1.00 29.05 A ATOM 299 CB ALA A 108 13.704 −24.934 55.3031.00 26.23 A ATOM 300 C ALA A 108 14.738 −25.194 53.039 1.00 29.49 AATOM 301 O ALA A 108 15.712 −25.930 52.874 1.00 29.41 A ATOM 302 N SER A109 13.725 −25.120 52.179 1.00 29.12 A ATOM 303 CA SER A 109 13.696−25.895 50.945 1.00 30.36 A ATOM 304 CB SER A 109 12.400 −25.612 50.1821.00 30.10 A ATOM 305 OG SER A 109 12.408 −26.258 48.926 1.00 34.79 AATOM 306 C SER A 109 14.891 −25.535 50.066 1.00 31.21 A ATOM 307 O SER A109 15.545 −26.417 49.504 1.00 33.48 A ATOM 308 N ASP A 110 15.172−24.239 49.947 1.00 29.99 A ATOM 309 CA ASP A 110 16.296 −23.776 49.1361.00 30.22 A ATOM 310 CB ASP A 110 16.341 −22.242 49.099 1.00 31.27 AATOM 311 CG ASP A 110 15.163 −21.635 48.345 1.00 32.67 A ATOM 312 OD1ASP A 110 15.010 −20.398 48.382 1.00 30.92 A ATOM 313 OD2 ASP A 11014.391 −22.388 47.713 1.00 35.49 A ATOM 314 C ASP A 110 17.617 −24.31149.682 1.00 29.51 A ATOM 315 O ASP A 110 18.492 −24.720 48.915 1.0027.25 A ATOM 316 N ALA A 111 17.757 −24.302 51.007 1.00 26.85 A ATOM 317CA ALA A 111 18.969 −24.791 51.661 1.00 26.33 A ATOM 318 CB ALA A 11118.901 −24.520 53.163 1.00 25.25 A ATOM 319 C ALA A 111 19.151 −26.28651.406 1.00 27.62 A ATOM 320 O ALA A 111 20.273 −26.768 51.235 1.0027.32 A ATOM 321 N LEU A 112 18.043 −27.020 51.385 1.00 26.45 A ATOM 322CA LEU A 112 18.101 −28.447 51.126 1.00 29.74 A ATOM 323 CB LEU A 11216.775 −29.116 51.514 1.00 28.30 A ATOM 324 CG LEU A 112 16.596 −29.28853.033 1.00 30.20 A ATOM 325 CD1 LEU A 112 15.130 −29.470 53.366 1.0030.47 A ATOM 326 CD2 LEU A 112 17.425 −30.474 53.528 1.00 28.57 A ATOM327 C LEU A 112 18.442 −28.706 49.655 1.00 30.58 A ATOM 328 O LEU A 11219.209 −29.618 49.352 1.00 28.99 A ATOM 329 N ASP A 113 17.893 −27.90548.745 1.00 30.56 A ATOM 330 CA ASP A 113 18.207 −28.090 47.327 1.0032.58 A ATOM 331 CB ASP A 113 17.479 −27.079 46.427 1.00 31.43 A ATOM332 CG ASP A 113 15.995 −27.366 46.278 1.00 31.87 A ATOM 333 OD1 ASP A113 15.590 −28.544 46.286 1.00 32.19 A ATOM 334 OD2 ASP A 113 15.229−26.396 46.119 1.00 34.18 A ATOM 335 C ASP A 113 19.707 −27.871 47.1591.00 32.53 A ATOM 336 O ASP A 113 20.382 −28.626 46.458 1.00 31.55 AATOM 337 N LYS A 114 20.221 −26.831 47.814 1.00 32.96 A ATOM 338 CA LYSA 114 21.636 −26.504 47.720 1.00 32.84 A ATOM 339 CB LYS A 114 21.956−25.259 48.547 1.00 37.08 A ATOM 340 CG LYS A 114 21.351 −23.983 47.9831.00 43.46 A ATOM 341 CD LYS A 114 21.838 −22.746 48.731 1.00 50.51 AATOM 342 CE LYS A 114 21.249 −21.466 48.137 1.00 52.99 A ATOM 343 NZ LYSA 114 21.695 −20.256 48.891 1.00 54.73 A ATOM 344 C LYS A 114 22.556−27.646 48.130 1.00 32.02 A ATOM 345 O LYS A 114 23.448 −28.024 47.3741.00 32.06 A ATOM 346 N ILE A 115 22.358 −28.197 49.323 1.00 30.95 AATOM 347 CA ILE A 115 23.216 −29.290 49.751 1.00 30.36 A ATOM 348 CB ILEA 115 23.006 −29.648 51.253 1.00 28.05 A ATOM 349 CG2 ILE A 115 21.703−30.404 51.451 1.00 29.39 A ATOM 350 CG1 ILE A 115 24.171 −30.515 51.7391.00 30.04 A ATOM 351 CD1 ILE A 115 25.540 −29.849 51.611 1.00 29.38 AATOM 352 C ILE A 115 22.984 −30.517 48.861 1.00 29.19 A ATOM 353 O ILE A115 23.897 −31.291 48.625 1.00 28.85 A ATOM 354 N ARG A 116 21.769−30.687 48.355 1.00 31.07 A ATOM 355 CA ARG A 116 21.495 −31.815 47.4741.00 35.04 A ATOM 356 CB ARG A 116 20.004 −31.900 47.124 1.00 37.53 AATOM 357 CG ARG A 116 19.712 −32.923 46.022 1.00 43.83 A ATOM 358 CD ARGA 116 18.230 −33.072 45.711 1.00 46.53 A ATOM 359 NE ARG A 116 17.525−33.854 46.724 1.00 50.14 A ATOM 360 CZ ARG A 116 16.234 −34.165 46.6660.00 49.23 A ATOM 361 NH1 ARG A 116 15.497 −33.759 45.642 0.00 49.63 AATOM 362 NH2 ARG A 116 15.679 −34.886 47.631 0.00 49.63 A ATOM 363 C ARGA 116 22.319 −31.656 46.190 1.00 35.93 A ATOM 364 O ARG A 116 22.931−32.608 45.720 1.00 35.09 A ATOM 365 N LEU A 117 22.342 −30.448 45.6341.00 35.02 A ATOM 366 CA LEU A 117 23.109 −30.192 44.416 1.00 35.33 AATOM 367 CB LEU A 117 22.848 −28.769 43.898 1.00 35.62 A ATOM 368 CG LEUA 117 21.416 −28.401 43.480 1.00 38.17 A ATOM 369 CD1 LEU A 117 21.396−26.984 42.917 1.00 38.15 A ATOM 370 CD2 LEU A 117 20.906 −29.380 42.4371.00 39.74 A ATOM 371 C LEU A 117 24.604 −30.372 44.688 1.00 35.09 AATOM 372 O LEU A 117 25.348 −30.862 43.838 1.00 32.38 A ATOM 373 N ILE A118 25.036 −29.976 45.880 1.00 33.96 A ATOM 374 CA ILE A 118 26.438−30.102 46.258 1.00 35.20 A ATOM 375 CB ILE A 118 26.719 −29.402 47.6061.00 35.56 A ATOM 376 CG2 ILE A 118 28.155 −29.686 48.058 1.00 33.74 AATOM 377 CG1 ILE A 118 26.481 −27.898 47.463 1.00 36.15 A ATOM 378 CD1ILE A 118 26.556 −27.127 48.782 1.00 37.01 A ATOM 379 C ILE A 118 26.856−31.566 46.373 1.00 35.27 A ATOM 380 O ILE A 118 28.006 −31.910 46.0871.00 34.03 A ATOM 381 N SER A 119 25.922 −32.422 46.784 1.00 35.20 AATOM 382 CA SER A 119 26.219 −33.842 46.939 1.00 36.19 A ATOM 383 CB SERA 119 25.043 −34.581 47.599 1.00 34.53 A ATOM 384 OG SER A 119 23.933−34.700 46.727 1.00 33.75 A ATOM 385 C SER A 119 26.532 −34.454 45.5781.00 37.53 A ATOM 386 O SER A 119 27.127 −35.529 45.494 1.00 36.37 AATOM 387 N LEU A 120 26.132 −33.761 44.515 1.00 38.69 A ATOM 388 CA LEUA 120 26.396 −34.233 43.157 1.00 41.79 A ATOM 389 CB LEU A 120 25.583−33.436 42.127 1.00 40.22 A ATOM 390 CG LEU A 120 24.063 −33.612 42.1061.00 42.57 A ATOM 391 CD1 LEU A 120 23.472 −32.764 40.985 1.00 42.67 AATOM 392 CD2 LEU A 120 23.710 −35.078 41.903 1.00 42.57 A ATOM 393 C LEUA 120 27.879 −34.096 42.824 1.00 42.04 A ATOM 394 O LEU A 120 28.428−34.900 42.074 1.00 44.04 A ATOM 395 N THR A 121 28.524 −33.079 43.3881.00 43.08 A ATOM 396 CA THR A 121 29.937 −32.834 43.127 1.00 44.56 AATOM 397 CB THR A 121 30.185 −31.371 42.751 1.00 44.06 A ATOM 398 OG1THR A 121 29.882 −30.534 43.875 1.00 44.67 A ATOM 399 CG2 THR A 12129.313 −30.969 41.574 1.00 43.68 A ATOM 400 C THR A 121 30.841 −33.15044.309 1.00 46.28 A ATOM 401 O THR A 121 32.061 −33.211 44.162 1.0045.91 A ATOM 402 N ASP A 122 30.244 −33.338 45.480 1.00 48.08 A ATOM 403CA ASP A 122 31.011 −33.630 46.683 1.00 49.23 A ATOM 404 CB ASP A 12230.869 −32.480 47.681 1.00 51.96 A ATOM 405 CG ASP A 122 31.901 −32.53548.787 1.00 54.29 A ATOM 406 OD1 ASP A 122 32.357 −33.650 49.122 1.0056.04 A ATOM 407 OD2 ASP A 122 32.246 −31.462 49.330 1.00 55.62 A ATOM408 C ASP A 122 30.494 −34.921 47.305 1.00 48.93 A ATOM 409 O ASP A 12229.392 −34.954 47.851 1.00 47.16 A ATOM 410 N ALA A 123 31.299 −35.97747.219 1.00 49.14 A ATOM 411 CA ALA A 123 30.930 −37.288 47.752 1.0049.93 A ATOM 412 CB ALA A 123 32.026 −38.305 47.431 1.00 50.62 A ATOM413 C ALA A 123 30.644 −37.295 49.253 1.00 50.38 A ATOM 414 O ALA A 12329.828 −38.086 49.726 1.00 52.10 A ATOM 415 N ASN A 124 31.308 −36.42350.004 1.00 50.62 A ATOM 416 CA ASN A 124 31.099 −36.372 51.451 1.0051.30 A ATOM 417 CB ASN A 124 32.447 −36.354 52.179 1.00 53.30 A ATOM418 CG ASN A 124 33.070 −37.733 52.283 1.00 55.50 A ATOM 419 OD1 ASN A124 33.395 −38.357 51.275 1.00 56.73 A ATOM 420 ND2 ASN A 124 33.234−38.218 53.511 1.00 55.50 A ATOM 421 C ASN A 124 30.253 −35.197 51.9361.00 49.31 A ATOM 422 O ASN A 124 30.179 −34.937 53.134 1.00 48.17 AATOM 423 N ALA A 125 29.605 −34.498 51.012 1.00 47.50 A ATOM 424 CA ALAA 125 28.786 −33.351 51.373 1.00 46.05 A ATOM 425 CB ALA A 125 28.023−32.857 50.152 1.00 43.99 A ATOM 426 C ALA A 125 27.811 −33.626 52.5211.00 44.71 A ATOM 427 O ALA A 125 27.620 −32.780 53.393 1.00 43.86 AATOM 428 N LEU A 126 27.209 −34.812 52.525 1.00 44.79 A ATOM 429 CA LEUA 126 26.224 −35.177 53.542 1.00 45.70 A ATOM 430 CB LEU A 126 25.107−35.990 52.883 1.00 45.39 A ATOM 431 CG LEU A 126 24.417 −35.278 51.7171.00 46.52 A ATOM 432 CD1 LEU A 126 23.404 −36.197 51.061 1.00 46.62 AATOM 433 CD2 LEU A 126 23.742 −34.014 52.233 1.00 47.81 A ATOM 434 C LEUA 126 26.747 −35.932 54.768 1.00 45.28 A ATOM 435 O LEU A 126 25.965−36.341 55.628 1.00 45.72 A ATOM 436 N ALA A 127 28.062 −36.096 54.8521.00 45.95 A ATOM 437 CA ALA A 127 28.698 −36.816 55.953 1.00 46.85 AATOM 438 CB ALA A 127 30.211 −36.789 55.771 1.00 46.17 A ATOM 439 C ALAA 127 28.344 −36.320 57.356 1.00 48.69 A ATOM 440 O ALA A 127 28.477−37.067 58.327 1.00 49.08 A ATOM 441 N GLY A 128 27.897 −35.071 57.4671.00 48.63 A ATOM 442 CA GLY A 128 27.562 −34.521 58.771 1.00 48.99 AATOM 443 C GLY A 128 26.153 −34.823 59.244 1.00 49.66 A ATOM 444 O GLY A128 25.841 −34.662 60.422 1.00 50.11 A ATOM 445 N ASN A 129 25.303−35.265 58.326 1.00 49.94 A ATOM 446 CA ASN A 129 23.920 −35.587 58.6421.00 50.71 A ATOM 447 CB ASN A 129 23.193 −34.323 59.121 1.00 50.81 AATOM 448 CG ASN A 129 21.807 −34.609 59.672 1.00 50.32 A ATOM 449 OD1ASN A 129 21.224 −33.775 60.366 1.00 50.86 A ATOM 450 ND2 ASN A 12921.269 −35.781 59.357 1.00 49.42 A ATOM 451 C ASN A 129 23.290 −36.13357.364 1.00 52.23 A ATOM 452 O ASN A 129 22.938 −35.378 56.460 1.0052.87 A ATOM 453 N GLU A 130 23.157 −37.453 57.303 1.00 53.42 A ATOM 454CA GLU A 130 22.608 −38.138 56.137 1.00 53.84 A ATOM 455 CB GLU A 13022.679 −39.650 56.354 1.00 57.50 A ATOM 456 CG GLU A 130 24.058 −40.16256.758 1.00 62.46 A ATOM 457 CD GLU A 130 25.093 −40.029 55.653 1.0065.01 A ATOM 458 OE1 GLU A 130 26.273 −40.351 55.910 1.00 66.73 A ATOM459 OE2 GLU A 130 24.731 −39.611 54.531 1.00 66.00 A ATOM 460 C GLU A130 21.179 −37.761 55.757 1.00 52.27 A ATOM 461 O GLU A 130 20.741−38.039 54.641 1.00 53.28 A ATOM 462 N ALA A 131 20.450 −37.128 56.6681.00 49.49 A ATOM 463 CA ALA A 131 19.065 −36.754 56.383 1.00 46.55 AATOM 464 CB ALA A 131 18.272 −36.690 57.688 1.00 47.39 A ATOM 465 C ALAA 131 18.899 −35.441 55.615 1.00 43.78 A ATOM 466 O ALA A 131 19.746−34.550 55.685 1.00 44.02 A ATOM 467 N LEU A 132 17.793 −35.347 54.8811.00 40.82 A ATOM 468 CA LEU A 132 17.431 −34.165 54.100 1.00 39.49 AATOM 469 CB LEU A 132 17.586 −34.438 52.602 1.00 38.45 A ATOM 470 CG LEUA 132 19.012 −34.686 52.111 1.00 39.04 A ATOM 471 CD1 LEU A 132 18.997−34.935 50.606 1.00 40.56 A ATOM 472 CD2 LEU A 132 19.880 −33.480 52.4461.00 37.22 A ATOM 473 C LEU A 132 15.966 −33.894 54.424 1.00 39.45 AATOM 474 O LEU A 132 15.069 −34.289 53.673 1.00 38.76 A ATOM 475 N THR A133 15.728 −33.211 55.541 1.00 37.29 A ATOM 476 CA THR A 133 14.369−32.944 55.990 1.00 34.35 A ATOM 477 CB THR A 133 13.972 −33.932 57.0961.00 35.57 A ATOM 478 OG1 THR A 133 14.756 −33.654 58.265 1.00 34.87 AATOM 479 CG2 THR A 133 14.229 −35.378 56.663 1.00 36.95 A ATOM 480 C THRA 133 14.141 −31.554 56.578 1.00 33.92 A ATOM 481 O THR A 133 15.079−30.800 56.842 1.00 33.54 A ATOM 482 N VAL A 134 12.864 −31.246 56.7821.00 35.11 A ATOM 483 CA VAL A 134 12.423 −30.007 57.404 1.00 35.04 AATOM 484 CB VAL A 134 11.559 −29.154 56.456 1.00 35.32 A ATOM 485 CG1VAL A 134 11.063 −27.920 57.188 1.00 34.33 A ATOM 486 CG2 VAL A 13412.370 −28.745 55.238 1.00 33.86 A ATOM 487 C VAL A 134 11.558 −30.46258.583 1.00 36.04 A ATOM 488 O VAL A 134 10.492 −31.053 58.385 1.0035.21 A ATOM 489 N LYS A 135 12.030 −30.211 59.801 1.00 36.08 A ATOM 490CA LYS A 135 11.301 −30.608 61.006 1.00 36.29 A ATOM 491 CB LYS A 13512.157 −31.558 61.846 1.00 36.25 A ATOM 492 CG LYS A 135 12.346 −32.93461.220 1.00 39.42 A ATOM 493 CD LYS A 135 13.328 −33.785 62.020 1.0040.39 A ATOM 494 CE LYS A 135 13.349 −35.230 61.522 1.00 40.38 A ATOM495 NZ LYS A 135 14.392 −36.039 62.230 1.00 39.55 A ATOM 496 C LYS A 13510.886 −29.403 61.847 1.00 37.12 A ATOM 497 O LYS A 135 11.712 −28.56462.214 1.00 36.87 A ATOM 498 N ILE A 136 9.598 −29.337 62.157 1.00 37.18A ATOM 499 CA ILE A 136 9.042 −28.237 62.929 1.00 37.49 A ATOM 500 CBILE A 136 7.940 −27.524 62.116 1.00 37.75 A ATOM 501 CG2 ILE A 136 7.233−26.486 62.983 1.00 37.38 A ATOM 502 CG1 ILE A 136 8.552 −26.896 60.8631.00 39.62 A ATOM 503 CD1 ILE A 136 7.542 −26.337 59.893 1.00 41.02 AATOM 504 C ILE A 136 8.439 −28.686 64.257 1.00 38.72 A ATOM 505 O ILE A136 7.727 −29.688 64.312 1.00 36.70 A ATOM 506 N LYS A 137 8.734 −27.95565.328 1.00 38.86 A ATOM 507 CA LYS A 137 8.153 −28.284 66.620 1.0042.04 A ATOM 508 CB LYS A 137 9.033 −29.267 67.416 1.00 45.10 A ATOM 509CG LYS A 137 10.511 −28.939 67.570 1.00 47.27 A ATOM 510 CD LYS A 13711.177 −30.056 68.373 1.00 49.53 A ATOM 511 CE LYS A 137 12.693 −29.93368.422 1.00 51.98 A ATOM 512 NZ LYS A 137 13.150 −28.727 69.162 1.0053.51 A ATOM 513 C LYS A 137 7.806 −27.068 67.472 1.00 42.49 A ATOM 514O LYS A 137 8.510 −26.056 67.482 1.00 40.52 A ATOM 515 N CYS A 138 6.687−27.183 68.172 1.00 42.67 A ATOM 516 CA CYS A 138 6.197 −26.120 69.0291.00 44.44 A ATOM 517 CB CYS A 138 4.678 −26.009 68.904 1.00 43.70 AATOM 518 SG CYS A 138 4.082 −25.900 67.213 1.00 45.30 A ATOM 519 C CYS A138 6.552 −26.414 70.474 1.00 43.99 A ATOM 520 O CYS A 138 6.560 −27.56570.901 1.00 43.91 A ATOM 521 N ASP A 139 6.855 −25.367 71.222 1.00 44.81A ATOM 522 CA ASP A 139 7.173 −25.519 72.628 1.00 46.03 A ATOM 523 CBASP A 139 8.653 −25.252 72.885 1.00 47.30 A ATOM 524 CG ASP A 139 9.105−25.785 74.225 1.00 49.47 A ATOM 525 OD1 ASP A 139 8.436 −25.483 75.2361.00 50.01 A ATOM 526 OD2 ASP A 139 10.123 −26.508 74.264 1.00 49.55 AATOM 527 C ASP A 139 6.319 −24.498 73.363 1.00 46.25 A ATOM 528 O ASP A139 6.811 −23.446 73.783 1.00 45.28 A ATOM 529 N ALA A 140 5.033 −24.81373.488 1.00 45.65 A ATOM 530 CA ALA A 140 4.074 −23.936 74.145 1.0048.13 A ATOM 531 CB ALA A 140 2.738 −24.662 74.321 1.00 48.04 A ATOM 532C ALA A 140 4.591 −23.460 75.492 1.00 48.45 A ATOM 533 O ALA A 140 4.490−22.282 75.824 1.00 49.34 A ATOM 534 N GLU A 141 5.155 −24.382 76.2601.00 50.21 A ATOM 535 CA GLU A 141 5.676 −24.057 77.578 1.00 51.71 AATOM 536 CB GLU A 141 6.163 −25.333 78.273 1.00 54.89 A ATOM 537 CG GLUA 141 5.019 −26.268 78.643 1.00 59.36 A ATOM 538 CD GLU A 141 5.485−27.577 79.249 1.00 63.14 A ATOM 539 OE1 GLU A 141 6.137 −28.371 78.5361.00 65.16 A ATOM 540 OE2 GLU A 141 5.195 −27.812 80.441 1.00 65.13 AATOM 541 C GLU A 141 6.779 −23.005 77.545 1.00 50.76 A ATOM 542 O GLU A141 6.847 −22.158 78.434 1.00 52.28 A ATOM 543 N ALA A 142 7.633 −23.04476.526 1.00 46.94 A ATOM 544 CA ALA A 142 8.708 −22.062 76.424 1.0043.37 A ATOM 545 CB ALA A 142 10.010 −22.745 76.021 1.00 44.14 A ATOM546 C ALA A 142 8.373 −20.942 75.438 1.00 41.63 A ATOM 547 O ALA A 1429.180 −20.040 75.220 1.00 42.01 A ATOM 548 N ALA A 143 7.179 −20.99774.853 1.00 38.59 A ATOM 549 CA ALA A 143 6.749 −19.983 73.893 1.0036.98 A ATOM 550 CB ALA A 143 6.676 −18.621 74.578 1.00 37.79 A ATOM 551C ALA A 143 7.683 −19.901 72.683 1.00 35.42 A ATOM 552 O ALA A 143 7.976−18.815 72.190 1.00 35.88 A ATOM 553 N LEU A 144 8.144 −21.048 72.2011.00 34.76 A ATOM 554 CA LEU A 144 9.048 −21.074 71.056 1.00 34.83 AATOM 555 CB LEU A 144 10.415 −21.623 71.475 1.00 35.13 A ATOM 556 CG LEUA 144 11.174 −20.954 72.622 1.00 36.82 A ATOM 557 CD1 LEU A 144 12.423−21.779 72.951 1.00 35.69 A ATOM 558 CD2 LEU A 144 11.548 −19.525 72.2331.00 35.89 A ATOM 559 C LEU A 144 8.529 −21.925 69.902 1.00 34.02 A ATOM560 O LEU A 144 7.810 −22.901 70.107 1.00 33.48 A ATOM 561 N LEU A 1458.899 −21.536 68.687 1.00 34.45 A ATOM 562 CA LEU A 145 8.540 −22.27967.486 1.00 33.67 A ATOM 563 CB LEU A 145 7.616 −21.452 66.582 1.0034.01 A ATOM 564 CG LEU A 145 7.163 −22.113 65.272 1.00 37.05 A ATOM 565CD1 LEU A 145 6.402 −23.404 65.587 1.00 38.01 A ATOM 566 CD2 LEU A 1456.281 −21.159 64.478 1.00 36.91 A ATOM 567 C LEU A 145 9.884 −22.53366.800 1.00 33.48 A ATOM 568 O LEU A 145 10.660 −21.600 66.587 1.0033.22 A ATOM 569 N HIS A 146 10.172 −23.794 66.487 1.00 33.54 A ATOM 570CA HIS A 146 11.436 −24.157 65.845 1.00 32.05 A ATOM 571 CB HIS A 14612.164 −25.239 66.652 1.00 32.73 A ATOM 1572 CG HIS A 146 12.458 −24.86568.073 1.00 30.80 A ATOM 573 CD2 HIS A 146 11.916 −25.301 69.237 1.0029.75 A ATOM 574 ND1 HIS A 146 13.464 −23.990 68.421 1.00 30.71 A ATOM575 CE1 HIS A 146 13.533 −23.909 69.740 1.00 33.06 A ATOM 576 NE2 HIS A146 12.605 −24.695 70.258 1.00 30.34 A ATOM 577 C HIS A 146 11.204−24.707 64.439 1.00 31.91 A ATOM 578 O HIS A 146 10.315 −25.527 64.2341.00 31.45 A ATOM 579 N VAL A 147 12.008 −24.251 63.482 1.00 29.51 AATOM 580 CA VAL A 147 11.927 −24.717 62.097 1.00 28.66 A ATOM 581 CB VALA 147 11.459 −23.605 61.137 1.00 28.09 A ATOM 582 CG1 VAL A 147 11.479−24.112 59.711 1.00 31.64 A ATOM 583 CG2 VAL A 147 10.066 −23.154 61.5101.00 26.76 A ATOM 584 C VAL A 147 13.349 −25.129 61.739 1.00 28.95 AATOM 585 O VAL A 147 14.211 −24.284 61.491 1.00 26.81 A ATOM 586 N THR A148 13.581 −26.438 61.730 1.00 29.47 A ATOM 587 CA THR A 148 14.900−26.988 61.462 1.00 30.68 A ATOM 588 CB THR A 148 15.342 −27.931 62.6121.00 33.73 A ATOM 589 OG1 THR A 148 15.276 −27.229 63.862 1.00 32.55 AATOM 590 CG2 THR A 148 16.773 −28.439 62.376 1.00 30.72 A ATOM 591 C THRA 148 14.992 −27.765 60.162 1.00 29.42 A ATOM 592 O THR A 148 14.126−28.576 59.836 1.00 29.39 A ATOM 593 N ASP A 149 16.061 −27.506 59.4241.00 29.96 A ATOM 594 CA ASP A 149 16.304 −28.192 58.170 1.00 29.42 AATOM 595 CB ASP A 149 16.159 −27.225 56.985 1.00 28.33 A ATOM 596 CG ASPA 149 17.258 −26.169 56.948 1.00 27.59 A ATOM 597 OD1 ASP A 149 18.431−26.534 56.737 1.00 26.90 A ATOM 598 OD2 ASP A 149 16.950 −24.971 57.1261.00 27.46 A ATOM 599 C ASP A 149 17.720 −28.741 58.228 1.00 28.78 AATOM 600 O ASP A 149 18.544 −28.274 59.016 1.00 27.96 A ATOM 601 N THR A150 17.987 −29.745 57.404 1.00 29.10 A ATOM 602 CA THR A 150 19.304−30.354 57.331 1.00 29.75 A ATOM 603 CB THR A 150 19.211 −31.901 57.4581.00 30.54 A ATOM 604 OG1 THR A 150 18.268 −32.422 56.508 1.00 30.80 AATOM 605 CG2 THR A 150 18.758 −32.282 58.870 1.00 30.06 A ATOM 606 C THRA 150 19.886 −29.948 55.980 1.00 31.93 A ATOM 607 O THR A 150 20.487−30.755 55.262 1.00 31.90 A ATOM 608 N GLY A 151 19.681 −28.673 55.6511.00 31.73 A ATOM 609 CA GLY A 151 20.166 −28.115 54.404 1.00 29.51 AATOM 610 C GLY A 151 21.633 −27.743 54.463 1.00 30.05 A ATOM 611 O GLY A151 22.372 −28.209 55.333 1.00 30.34 A ATOM 612 N VAL A 152 22.059−26.880 53.549 1.00 28.78 A ATOM 613 CA VAL A 152 23.460 −26.488 53.4901.00 26.43 A ATOM 614 CB VAL A 152 23.722 −25.600 52.256 1.00 25.38 AATOM 615 CG1 VAL A 152 23.041 −24.258 52.421 1.00 23.19 A ATOM 616 CG2VAL A 152 25.222 −25.439 52.039 1.00 22.48 A ATOM 617 C VAL A 152 23.978−25.779 54.746 1.00 28.64 A ATOM 618 O VAL A 152 25.161 −25.858 55.0621.00 27.06 A ATOM 619 N GLY A 153 23.095 −25.098 55.470 1.00 27.77 AATOM 620 CA GLY A 153 23.533 −24.394 56.660 1.00 26.50 A ATOM 621 C GLYA 153 24.267 −23.115 56.293 1.00 29.07 A ATOM 622 O GLY A 153 24.364−22.763 55.113 1.00 29.40 A ATOM 623 N MET A 154 24.785 −22.419 57.3021.00 27.36 A ATOM 624 CA MET A 154 25.507 −21.171 57.084 1.00 28.29 AATOM 625 CB MET A 154 24.598 −19.961 57.358 1.00 27.33 A ATOM 626 CG META 154 23.533 −19.684 56.299 1.00 31.21 A ATOM 627 SD MET A 154 22.490−18.255 56.745 1.00 29.78 A ATOM 628 CE MET A 154 21.274 −19.072 57.7231.00 26.73 A ATOM 629 C MET A 154 26.728 −21.054 57.980 1.00 26.97 AATOM 630 O MET A 154 26.691 −21.446 59.142 1.00 27.36 A ATOM 631 N THR A155 27.804 −20.504 57.433 1.00 27.79 A ATOM 632 CA THR A 155 29.023−20.293 58.196 1.00 29.31 A ATOM 633 CB THR A 155 30.237 −20.064 57.2851.00 29.15 A ATOM 634 OG1 THR A 155 30.090 −18.806 56.610 1.00 30.37 AATOM 635 CG2 THR A 155 30.358 −21.193 56.254 1.00 27.57 A ATOM 636 C THRA 155 28.809 −19.013 58.991 1.00 30.74 A ATOM 637 O THR A 155 27.861−18.264 58.737 1.00 30.95 A ATOM 638 N ARG A 156 29.695 −18.752 59.9431.00 31.93 A ATOM 639 CA ARG A 156 29.582 −17.549 60.747 1.00 31.40 AATOM 640 CB ARG A 156 30.781 −17.429 61.679 1.00 33.12 A ATOM 641 CG ARGA 156 30.804 −16.141 62.466 1.00 36.80 A ATOM 642 CD ARG A 156 31.986−16.079 63.416 1.00 37.87 A ATOM 643 NE ARG A 156 32.005 −14.810 64.1321.00 42.31 A ATOM 644 CZ ARG A 156 32.198 −13.633 63.550 1.00 43.07 AATOM 645 NH1 ARG A 156 32.396 −13.562 62.244 1.00 45.16 A ATOM 646 NH2ARG A 156 32.178 −12.525 64.271 1.00 47.44 A ATOM 647 C ARG A 156 29.491−16.313 59.854 1.00 30.98 A ATOM 648 O ARG A 156 28.628 −15.446 60.0571.00 29.50 A ATOM 649 N ALA A 157 30.376 −16.238 58.862 1.00 28.79 AATOM 650 CA ALA A 157 30.405 −15.094 57.949 1.00 29.13 A ATOM 651 CB ALAA 157 31.579 −15.216 56.977 1.00 28.16 A ATOM 652 C ALA A 157 29.107−14.925 57.167 1.00 27.42 A ATOM 653 O ALA A 157 28.700 −13.810 56.8731.00 27.13 A ATOM 654 N GLU A 158 28.467 −16.031 56.821 1.00 28.87 AATOM 655 CA GLU A 158 27.217 −15.973 56.076 1.00 30.24 A ATOM 656 CB GLUA 158 26.904 −17.343 55.490 1.00 31.06 A ATOM 657 CG GLU A 158 27.982−17.833 54.535 1.00 36.51 A ATOM 658 CD GLU A 158 27.600 −19.120 53.8491.00 36.81 A ATOM 659 OE1 GLU A 158 27.342 −20.120 54.549 1.00 38.69 AATOM 660 OE2 GLU A 158 27.555 −19.130 52.606 1.00 43.45 A ATOM 661 C GLUA 158 26.061 −15.504 56.960 1.00 31.29 A ATOM 662 O GLU A 158 25.158−14.804 56.494 1.00 30.64 A ATOM 663 N LEU A 159 26.087 −15.900 58.2311.00 29.36 A ATOM 664 CA LEU A 159 25.046 −15.494 59.165 1.00 31.34 AATOM 665 CB LEU A 159 25.297 −16.107 60.549 1.00 28.12 A ATOM 666 CG LEUA 159 24.976 −17.599 60.666 1.00 28.89 A ATOM 667 CD1 LEU A 159 25.520−18.155 61.967 1.00 27.34 A ATOM 668 CD2 LEU A 159 23.469 −17.804 60.5761.00 24.98 A ATOM 669 C LEU A 159 25.067 −13.977 59.249 1.00 31.03 AATOM 670 O LEU A 159 24.023 −13.321 59.223 1.00 32.32 A ATOM 671 N VAL A160 26.275 −13.432 59.326 1.00 30.83 A ATOM 672 CA VAL A 160 26.480−11.996 59.410 1.00 31.27 A ATOM 673 CB VAL A 160 27.953 −11.667 59.7321.00 30.71 A ATOM 674 CG1 VAL A 160 28.198 −10.155 59.581 1.00 29.76 AATOM 675 CG2 VAL A 160 28.288 −12.126 61.145 1.00 28.92 A ATOM 676 C VALA 160 26.108 −11.271 58.121 1.00 32.45 A ATOM 677 O VAL A 160 25.329−10.317 58.137 1.00 34.57 A ATOM 678 N ALA A 161 26.667 −11.727 57.0071.00 30.48 A ATOM 679 CA ALA A 161 26.420 −11.093 55.719 1.00 31.23 AATOM 680 CB ALA A 161 27.477 −11.555 54.705 1.00 30.35 A ATOM 681 C ALAA 161 25.022 −11.292 55.131 1.00 30.55 A ATOM 682 O ALA A 161 24.348−10.322 54.785 1.00 29.64 A ATOM 683 N ASN A 162 24.604 −12.548 55.0121.00 30.70 A ATOM 684 CA ASN A 162 23.310 −12.901 54.429 1.00 31.28 AATOM 685 CB ASN A 162 23.180 −14.423 54.343 1.00 30.39 A ATOM 686 CG ASNA 162 24.206 −15.047 53.414 1.00 32.65 A ATOM 687 OD1 ASN A 162 25.338−14.580 53.319 1.00 34.15 A ATOM 688 ND2 ASN A 162 23.818 −16.120 52.7411.00 32.64 A ATOM 689 C ASN A 162 22.085 −12.346 55.147 1.00 31.78 AATOM 690 O ASN A 162 21.085 −12.019 54.504 1.00 31.02 A ATOM 691 N LEU A163 22.154 −12.256 56.472 1.00 29.42 A ATOM 692 CA LEU A 163 21.025−11.757 57.250 1.00 30.07 A ATOM 693 CB LEU A 163 20.817 −12.631 58.4991.00 29.43 A ATOM 694 CG LEU A 163 20.621 −14.146 58.302 1.00 29.68 AATOM 695 CD1 LEU A 163 20.443 −14.820 59.659 1.00 27.61 A ATOM 696 CD2LEU A 163 19.405 −14.409 57.411 1.00 28.58 A ATOM 697 C LEU A 163 21.180−10.298 57.674 1.00 29.99 A ATOM 698 O LEU A 163 20.192 −9.591 57.8451.00 27.85 A ATOM 699 N GLY A 164 22.420 −9.844 57.828 1.00 29.36 A ATOM700 CA GLY A 164 22.650 −8.480 58.277 1.00 29.63 A ATOM 701 C GLY A 16422.848 −7.419 57.214 1.00 30.34 A ATOM 702 O GLY A 164 22.791 −6.22557.514 1.00 30.77 A ATOM 703 N THR A 165 23.075 −7.838 55.974 1.00 28.12A ATOM 704 CA THR A 165 23.288 −6.888 54.893 1.00 29.13 A ATOM 705 CBTHR A 165 24.783 −6.771 54.521 1.00 29.76 A ATOM 706 OG1 THR A 16525.119 −7.813 53.595 1.00 26.17 A ATOM 707 CG2 THR A 165 25.670 −6.91055.764 1.00 28.83 A ATOM 708 C THR A 165 22.582 −7.342 53.630 1.00 30.18A ATOM 709 O THR A 165 22.001 −8.423 53.573 1.00 27.64 A ATOM 710 N ILEA 166 22.655 −6.500 52.611 1.00 34.77 A ATOM 711 CA ILE A 166 22.080−6.818 51.320 1.00 40.37 A ATOM 712 CB ILE A 166 21.673 −5.543 50.5711.00 42.39 A ATOM 713 CG2 ILE A 166 21.394 −5.855 49.093 1.00 42.55 AATOM 714 CG1 ILE A 166 20.444 −4.935 51.258 1.00 43.15 A ATOM 715 CD1ILE A 166 19.822 −3.800 50.491 1.00 48.78 A ATOM 716 C ILE A 166 23.200−7.541 50.587 1.00 44.58 A ATOM 717 O ILE A 166 24.079 −6.920 49.9861.00 45.27 A ATOM 718 N ALA A 167 23.177 −8.865 50.675 1.00 48.19 A ATOM719 CA ALA A 167 24.197 −9.693 50.057 1.00 52.05 A ATOM 720 CB ALA A 16724.266 −11.040 50.776 1.00 50.60 A ATOM 721 C ALA A 167 23.977 −9.90348.563 1.00 54.42 A ATOM 722 O ALA A 167 24.671 −9.311 47.737 1.00 55.92A ATOM 723 N LYS A 168 23.000 −10.737 48.222 1.00 57.35 A ATOM 724 CALYS A 168 22.700 −11.053 46.829 1.00 59.01 A ATOM 725 CB LYS A 16821.674 −12.188 46.782 1.00 61.02 A ATOM 726 CG LYS A 168 22.122 −13.42647.546 1.00 63.34 A ATOM 727 CD LYS A 168 21.095 −14.549 47.503 1.0065.33 A ATOM 728 CE LYS A 168 21.582 −15.744 48.320 1.00 66.58 A ATOM729 NZ LYS A 168 20.634 −16.891 48.295 1.00 67.20 A ATOM 730 C LYS A 16822.205 −9.865 46.006 1.00 59.60 A ATOM 731 O LYS A 168 21.591 −8.93946.538 1.00 59.93 A ATOM 732 N SER A 169 22.480 −9.902 44.703 1.00 58.96A ATOM 733 CA SER A 169 22.064 −8.838 43.788 1.00 58.85 A ATOM 734 CBSER A 169 22.587 −9.110 42.373 1.00 61.11 A ATOM 735 OG SER A 169 24.004−9.199 42.343 1.00 67.08 A ATOM 736 C SER A 169 20.544 −8.749 43.7391.00 56.48 A ATOM 737 O SER A 169 19.975 −7.671 43.564 1.00 54.95 A ATOM738 N GLY A 170 19.894 −9.895 43.890 1.00 54.51 A ATOM 739 CA GLY A 17018.446 −9.932 43.853 1.00 52.69 A ATOM 740 C GLY A 170 17.779 −9.11444.942 1.00 51.35 A ATOM 741 O GLY A 170 16.696 −8.566 44.730 1.00 52.46A ATOM 742 N THR A 171 18.416 −9.025 46.108 1.00 48.81 A ATOM 743 CA THRA 171 17.845 −8.272 47.218 1.00 46.14 A ATOM 744 CB THR A 171 18.731−8.350 48.471 1.00 44.42 A ATOM 745 OG1 THR A 171 18.952 −9.723 48.8141.00 44.07 A ATOM 746 CG2 THR A 171 18.052 −7.648 49.641 1.00 42.55 AATOM 747 C THR A 171 17.658 −6.810 46.843 1.00 45.48 A ATOM 748 O THR A171 16.618 −6.214 47.128 1.00 43.21 A ATOM 749 N SER A 172 18.670 −6.24146.198 1.00 46.26 A ATOM 750 CA SER A 172 18.620 −4.848 45.774 1.0047.43 A ATOM 751 CB SER A 172 19.960 −4.443 45.154 1.00 49.11 A ATOM 752OG SER A 172 19.969 −3.067 44.815 1.00 53.00 A ATOM 753 C SER A 17217.484 −4.640 44.765 1.00 47.57 A ATOM 754 O SER A 172 16.801 −3.61444.784 1.00 46.65 A ATOM 755 N ALA A 173 17.284 −5.620 43.887 1.00 47.00A ATOM 756 CA ALA A 173 16.225 −5.538 42.890 1.00 47.42 A ATOM 757 CBALA A 173 16.335 −6.704 41.909 1.00 46.58 A ATOM 758 C ALA A 173 14.877−5.574 43.610 1.00 46.92 A ATOM 759 O ALA A 173 13.989 −4.765 43.3301.00 46.07 A ATOM 760 N PHE A 174 14.732 −6.514 44.542 1.00 46.28 A ATOM761 CA PHE A 174 13.494 −6.635 45.300 1.00 46.39 A ATOM 762 CB PHE A 17413.621 −7.703 46.391 1.00 44.34 A ATOM 763 CG PHE A 174 12.585 −7.58047.473 1.00 40.11 A ATOM 764 CD1 PHE A 174 12.841 −6.828 48.615 1.0037.31 A ATOM 765 CD2 PHE A 174 11.333 −8.171 47.326 1.00 39.00 A ATOM766 CE1 PHE A 174 11.864 −6.664 49.596 1.00 39.89 A ATOM 767 CE2 PHE A174 10.345 −8.014 48.300 1.00 37.97 A ATOM 768 CZ PHE A 174 10.608−7.260 49.437 1.00 39.56 A ATOM 769 C PHE A 174 13.092 −5.307 45.9391.00 47.35 A ATOM 770 O PHE A 174 11.918 −4.938 45.928 1.00 47.86 A ATOM771 N LEU A 175 14.062 −4.593 46.500 1.00 47.40 A ATOM 772 CA LEU A 17513.773 −3.311 47.132 1.00 48.97 A ATOM 773 CB LEU A 175 15.045 −2.71247.733 1.00 49.01 A ATOM 774 CG LEU A 175 15.582 −3.390 48.994 1.0050.83 A ATOM 775 CD1 LEU A 175 16.863 −2.695 49.445 1.00 50.32 A ATOM776 CD2 LEU A 175 14.528 −3.326 50.094 1.00 52.00 A ATOM 777 C LEU A 17513.157 −2.321 46.149 1.00 50.06 A ATOM 778 O LEU A 175 12.259 −1.55746.508 1.00 49.84 A ATOM 779 N ASN A 176 13.644 −2.331 44.912 1.00 50.71A ATOM 780 CA ASN A 176 13.127 −1.436 43.888 1.00 50.81 A ATOM 781 CBASN A 176 14.076 −1.391 42.690 1.00 53.31 A ATOM 782 CG ASN A 176 15.414−0.770 43.031 1.00 55.99 A ATOM 783 OD1 ASN A 176 15.482 0.363 43.5151.00 57.64 A ATOM 784 ND2 ASN A 176 16.490 −1.506 42.778 1.00 57.47 AATOM 785 C ASN A 176 11.749 −1.891 43.426 1.00 50.13 A ATOM 786 O ASN A176 10.809 −1.097 43.374 1.00 50.94 A ATOM 787 N ALA A 177 11.630 −3.17143.091 1.00 48.58 A ATOM 788 CA ALA A 177 10.361 −3.713 42.631 1.0046.44 A ATOM 789 CB ALA A 177 10.533 −5.166 42.211 1.00 45.93 A ATOM 790C ALA A 177 9.313 −3.600 43.729 1.00 46.54 A ATOM 791 O ALA A 177 8.149−3.305 43.458 1.00 44.04 A ATOM 792 N MET A 178 9.734 −3.823 44.970 1.0046.54 A ATOM 793 CA MET A 178 8.825 −3.747 46.108 1.00 47.79 A ATOM 794CB MET A 178 9.555 −4.116 47.401 1.00 44.99 A ATOM 795 CG MET A 1788.658 −4.159 48.622 1.00 43.88 A ATOM 796 SD MET A 178 7.324 −5.34248.411 1.00 41.19 A ATOM 797 CE MET A 178 6.603 −5.337 50.037 1.00 42.69A ATOM 798 C MET A 178 8.230 −2.353 46.241 1.00 49.94 A ATOM 799 O MET A178 7.016 −2.196 46.384 1.00 49.65 A ATOM 800 N THR A 179 9.089 −1.34146.181 1.00 52.61 A ATOM 801 CA THR A 179 8.653 0.046 46.300 1.00 55.88A ATOM 802 CB THR A 179 9.864 0.998 46.418 1.00 56.76 A ATOM 803 OG1 THRA 179 10.748 0.786 45.311 1.00 57.84 A ATOM 804 CG2 THR A 179 10.6160.746 47.720 1.00 56.79 A ATOM 805 C THR A 179 7.789 0.491 45.121 1.0057.35 A ATOM 806 O THR A 179 6.826 1.237 45.299 1.00 58.41 A ATOM 807 NGLU A 180 8.131 0.038 43.919 1.00 58.61 A ATOM 808 CA GLU A 180 7.3700.402 42.729 1.00 60.56 A ATOM 809 CB GLU A 180 8.141 0.029 41.461 1.0061.60 A ATOM 810 CG GLU A 180 9.338 0.920 41.176 1.00 65.95 A ATOM 811CD GLU A 180 9.931 0.676 39.801 1.00 68.00 A ATOM 812 OE1 GLU A 18010.894 1.383 39.430 1.00 69.00 A ATOM 813 OE2 GLU A 180 9.431 −0.22339.089 1.00 70.25 A ATOM 814 C GLU A 180 5.994 −0.255 42.696 1.00 61.73A ATOM 815 O GLU A 180 5.068 0.261 42.071 1.00 62.24 A ATOM 816 N ALA A181 5.863 −1.396 43.362 1.00 61.63 A ATOM 817 CA ALA A 181 4.591 −2.10543.400 1.00 61.69 A ATOM 818 CB ALA A 181 4.829 −3.603 43.531 1.00 60.74A ATOM 819 C ALA A 181 3.762 −1.606 44.574 1.00 62.57 A ATOM 820 O ALA A181 2.583 −1.284 44.425 1.00 61.72 A ATOM 821 N GLN A 182 4.399 −1.53845.739 1.00 64.18 A ATOM 822 CA GLN A 182 3.749 −1.100 46.970 1.00 65.69A ATOM 823 CB GLN A 182 4.733 −1.233 48.136 1.00 65.51 A ATOM 824 CG GLNA 182 4.129 −1.023 49.506 1.00 66.97 A ATOM 825 CD GLN A 182 4.866−1.793 50.585 1.00 67.65 A ATOM 826 OE1 GLN A 182 4.906 −3.021 50.5641.00 68.71 A ATOM 827 NE2 GLN A 182 5.453 −1.075 51.536 1.00 69.42 AATOM 828 C GLN A 182 3.241 0.333 46.852 1.00 66.94 A ATOM 829 O GLN A182 2.449 0.796 47.675 1.00 66.83 A ATOM 830 N GLU A 183 3.695 1.02445.813 1.00 68.18 A ATOM 831 CA GLU A 183 3.289 2.400 45.565 1.00 69.69A ATOM 832 CB GLU A 183 4.479 3.209 45.038 1.00 70.89 A ATOM 833 CG GLUA 183 4.120 4.592 44.522 1.00 73.65 A ATOM 834 CD GLU A 183 5.333 5.37044.044 1.00 75.88 A ATOM 835 OE1 GLU A 183 5.148 6.458 43.453 1.00 76.43A ATOM 836 OE2 GLU A 183 6.470 4.896 44.264 1.00 76.85 A ATOM 837 C GLUA 183 2.147 2.445 44.554 1.00 69.18 A ATOM 838 O GLU A 183 1.225 3.25244.680 1.00 69.16 A ATOM 839 N ASP A 184 2.212 1.567 43.557 1.00 68.85 AATOM 840 CA ASP A 184 1.194 1.506 42.514 1.00 68.71 A ATOM 841 CB ASP A184 1.838 1.106 41.186 1.00 70.49 A ATOM 842 CG ASP A 184 2.748 2.18240.637 1.00 73.35 A ATOM 843 OD1 ASP A 184 2.278 3.327 40.471 1.00 75.84A ATOM 844 OD2 ASP A 184 3.931 1.888 40.367 1.00 75.42 A ATOM 845 C ASPA 184 0.031 0.563 42.830 1.00 67.37 A ATOM 846 O ASP A 184 −0.901 0.43242.036 1.00 67.70 A ATOM 847 N GLY A 185 0.089 −0.097 43.983 1.00 65.71A ATOM 848 CA GLY A 185 −0.986 −0.994 44.373 1.00 63.02 A ATOM 849 C GLYA 185 −0.868 −2.456 43.975 1.00 60.09 A ATOM 850 O GLY A 185 −1.689−3.274 44.396 1.00 60.36 A ATOM 851 N GLN A 186 0.136 −2.793 43.169 1.0056.39 A ATOM 852 CA GLN A 186 0.335 −4.175 42.730 1.00 53.38 A ATOM 853CB GLN A 186 1.484 −4.234 41.724 1.00 54.26 A ATOM 854 CG GLN A 1861.215 −3.431 40.458 1.00 57.47 A ATOM 855 CD GLN A 186 2.435 −3.31739.566 1.00 58.77 A ATOM 856 OE1 GLN A 186 3.010 −4.322 39.147 1.0060.77 A ATOM 857 NE2 GLN A 186 2.838 −2.086 39.272 1.00 58.40 A ATOM 858C GLN A 186 0.623 −5.093 43.922 1.00 49.70 A ATOM 859 O GLN A 186 1.187−4.654 44.921 1.00 49.64 A ATOM 860 N SER A 187 0.229 −6.361 43.818 1.0044.90 A ATOM 861 CA SER A 187 0.443 −7.315 44.908 1.00 41.67 A ATOM 862CB SER A 187 −0.162 −8.672 44.557 1.00 39.85 A ATOM 863 OG SER A 187−0.242 −9.489 45.712 1.00 40.74 A ATOM 864 C SER A 187 1.933 −7.46845.215 1.00 38.72 A ATOM 865 O SER A 187 2.762 −7.488 44.311 1.00 38.04A ATOM 866 N THR A 188 2.260 −7.587 46.497 1.00 37.99 A ATOM 867 CA THRA 188 3.651 −7.688 46.926 1.00 38.34 A ATOM 868 CB THR A 188 3.994−6.564 47.917 1.00 37.82 A ATOM 869 OG1 THR A 188 3.044 −6.582 48.9881.00 37.92 A ATOM 870 CG2 THR A 188 3.969 −5.209 47.230 1.00 35.82 AATOM 871 C THR A 188 4.102 −8.996 47.572 1.00 36.79 A ATOM 872 O THR A188 5.296 −9.275 47.605 1.00 38.88 A ATOM 873 N SER A 189 3.177 −9.79048.096 1.00 35.49 A ATOM 874 CA SER A 189 3.581 −11.041 48.733 1.0034.95 A ATOM 875 CB SER A 189 2.362 −11.813 49.269 1.00 35.44 A ATOM 876OG SER A 189 1.528 −12.301 48.232 1.00 35.99 A ATOM 877 C SER A 1894.370 −11.910 47.755 1.00 35.51 A ATOM 878 O SER A 189 5.301 −12.61548.155 1.00 34.15 A ATOM 879 N ALA A 190 4.014 −11.838 46.473 1.00 33.02A ATOM 880 CA ALA A 190 4.700 −12.624 45.453 1.00 33.42 A ATOM 881 CBALA A 190 3.964 −12.531 44.120 1.00 35.18 A ATOM 882 C ALA A 190 6.146−12.176 45.282 1.00 33.93 A ATOM 883 O ALA A 190 7.020 −13.001 45.0391.00 33.38 A ATOM 884 N LEU A 191 6.393 −10.874 45.407 1.00 33.98 A ATOM885 CA LEU A 191 7.744 −10.339 45.271 1.00 35.42 A ATOM 886 CB LEU A 1917.742 −8.809 45.367 1.00 35.78 A ATOM 887 CG LEU A 191 7.137 −8.01844.208 1.00 38.28 A ATOM 888 CD1 LEU A 191 7.349 −6.517 44.448 1.0036.74 A ATOM 889 CD2 LEU A 191 7.798 −8.443 42.906 1.00 37.70 A ATOM 890C LEU A 191 8.641 −10.902 46.366 1.00 35.52 A ATOM 891 O LEU A 191 9.766−11.342 46.100 1.00 35.56 A ATOM 892 N ILE A 192 8.133 −10.880 47.5951.00 34.69 A ATOM 893 CA ILE A 192 8.871 −11.387 48.743 1.00 33.73 AATOM 894 CB ILE A 192 8.020 −11.254 50.034 1.00 33.89 A ATOM 895 CG2 ILEA 192 8.722 −11.924 51.208 1.00 31.29 A ATOM 896 CG1 ILE A 192 7.789−9.767 50.332 1.00 32.32 A ATOM 897 CD1 ILE A 192 6.801 −9.494 51.4351.00 33.13 A ATOM 898 C ILE A 192 9.260 −12.843 48.495 1.00 33.70 A ATOM899 O ILE A 192 10.419 −13.210 48.639 1.00 32.55 A ATOM 900 N GLY A 1938.289 −13.666 48.109 1.00 34.34 A ATOM 901 CA GLY A 193 8.585 −15.05847.827 1.00 33.70 A ATOM 902 C GLY A 193 9.557 −15.196 46.664 1.00 36.77A ATOM 903 O GLY A 193 10.534 −15.948 46.750 1.00 36.23 A ATOM 904 N GLNA 194 9.301 −14.462 45.580 1.00 36.72 A ATOM 905 CA GLN A 194 10.150−14.511 44.390 1.00 37.60 A ATOM 906 CB GLN A 194 9.680 −13.498 43.3411.00 39.57 A ATOM 907 CG GLN A 194 8.397 −13.862 42.616 1.00 43.85 AATOM 908 CD GLN A 194 8.021 −12.841 41.548 1.00 46.63 A ATOM 909 OE1 GLNA 194 6.938 −12.909 40.961 1.00 49.46 A ATOM 910 NE2 GLN A 194 8.919−11.891 41.290 1.00 45.31 A ATOM 911 C GLN A 194 11.619 −14.238 44.6841.00 37.50 A ATOM 912 O GLN A 194 12.499 −14.964 44.225 1.00 37.05 AATOM 913 N PHE A 195 11.878 −13.174 45.432 1.00 36.67 A ATOM 914 CA PHEA 195 13.242 −12.801 45.765 1.00 37.67 A ATOM 915 CB PHE A 195 13.341−11.276 45.816 1.00 38.15 A ATOM 916 CG PHE A 195 13.253 −10.637 44.4611.00 41.48 A ATOM 917 CD1 PHE A 195 14.399 −10.435 43.698 1.00 42.51 AATOM 918 CD2 PHE A 195 12.016 −10.301 43.915 1.00 43.12 A ATOM 919 CE1PHE A 195 14.316 −9.909 42.406 1.00 45.11 A ATOM 920 CE2 PHE A 19511.916 −9.776 42.626 1.00 44.12 A ATOM 921 CZ PHE A 195 13.069 −9.57941.867 1.00 45.02 A ATOM 922 C PHE A 195 13.754 −13.444 47.055 1.0035.33 A ATOM 923 O PHE A 195 14.855 −13.154 47.507 1.00 34.49 A ATOM 924N GLY A 196 12.948 −14.338 47.622 1.00 33.40 A ATOM 925 CA GLY A 19613.330 −15.035 48.838 1.00 33.29 A ATOM 926 C GLY A 196 13.854 −14.18849.985 1.00 32.03 A ATOM 927 O GLY A 196 14.838 −14.557 50.626 1.0030.59 A ATOM 928 N VAL A 197 13.192 −13.067 50.259 1.00 29.74 A ATOM 929CA VAL A 197 13.604 −12.185 51.348 1.00 28.13 A ATOM 930 CB VAL A 19713.693 −10.712 50.872 1.00 28.77 A ATOM 931 CG1 VAL A 197 14.739 −10.57949.771 1.00 30.35 A ATOM 932 CG2 VAL A 197 12.336 −10.244 50.372 1.0029.75 A ATOM 933 C VAL A 197 12.604 −12.265 52.501 1.00 27.80 A ATOM 934O VAL A 197 12.524 −11.358 53.327 1.00 26.49 A ATOM 935 N GLY A 19811.863 −13.367 52.557 1.00 25.15 A ATOM 936 CA GLY A 198 10.848 −13.54653.582 1.00 24.56 A ATOM 937 C GLY A 198 11.297 −13.804 55.013 1.0025.45 A ATOM 938 O GLY A 198 10.482 −13.725 55.931 1.00 25.76 A ATOM 939N PHE A 199 12.575 −14.110 55.216 1.00 25.68 A ATOM 940 CA PHE A 19913.088 −14.369 56.561 1.00 23.94 A ATOM 941 CB PHE A 199 14.619 −14.47756.534 1.00 22.33 A ATOM 942 CG PHE A 199 15.250 −14.453 57.898 1.0023.33 A ATOM 943 CD1 PHE A 199 15.252 −15.590 58.702 1.00 22.87 A ATOM944 CD2 PHE A 199 15.821 −13.277 58.393 1.00 24.46 A ATOM 945 CE1 PHE A199 15.816 −15.560 59.982 1.00 24.30 A ATOM 946 CE2 PHE A 199 16.385−13.240 59.670 1.00 26.24 A ATOM 947 CZ PHE A 199 16.381 −14.390 60.4651.00 24.40 A ATOM 948 C PHE A 199 12.681 −13.279 57.559 1.00 25.64 AATOM 949 O PHE A 199 12.303 −13.567 58.697 1.00 27.18 A ATOM 950 N TYR A200 12.750 −12.026 57.127 1.00 25.95 A ATOM 951 CA TYR A 200 12.410−10.908 57.994 1.00 25.91 A ATOM 952 CB TYR A 200 12.806 −9.598 57.3131.00 25.62 A ATOM 953 CG TYR A 200 14.279 −9.534 56.957 1.00 26.90 AATOM 954 CD1 TYR A 200 15.263 −9.518 57.948 1.00 24.43 A ATOM 955 CE1TYR A 200 16.624 −9.494 57.614 1.00 27.21 A ATOM 956 CD2 TYR A 20014.689 −9.524 55.627 1.00 27.49 A ATOM 957 CE2 TYR A 200 16.034 −9.50055.287 1.00 27.42 A ATOM 958 CZ TYR A 200 16.997 −9.485 56.278 1.0028.42 A ATOM 959 OH TYR A 200 18.326 −9.462 55.916 1.00 28.80 A ATOM 960C TYR A 200 10.938 −10.845 58.432 1.00 26.48 A ATOM 961 O TYR A 20010.624 −10.227 59.455 1.00 24.30 A ATOM 962 N SER A 201 10.039 −11.48857.687 1.00 24.40 A ATOM 963 CA SER A 201 8.624 −11.449 58.065 1.0025.63 A ATOM 964 CB SER A 201 7.736 −12.040 56.961 1.00 23.58 A ATOM 965OG SER A 201 7.904 −13.439 56.842 1.00 24.58 A ATOM 966 C SER A 2018.395 −12.193 59.379 1.00 25.70 A ATOM 967 O SER A 201 7.312 −12.12959.963 1.00 26.74 A ATOM 968 N ALA A 202 9.423 −12.898 59.841 1.00 25.08A ATOM 969 CA ALA A 202 9.332 −13.625 61.094 1.00 24.60 A ATOM 970 CBALA A 202 10.618 −14.416 61.347 1.00 25.15 A ATOM 971 C ALA A 202 9.089−12.640 62.236 1.00 24.58 A ATOM 972 O ALA A 202 8.549 −13.009 63.2771.00 25.71 A ATOM 973 N PHE A 203 9.485 −11.386 62.040 1.00 25.13 A ATOM974 CA PHE A 203 9.298 −10.372 63.077 1.00 26.24 A ATOM 975 CB PHE A 20310.179 −9.153 62.794 1.00 27.79 A ATOM 976 CG PHE A 203 11.633 −9.37663.141 1.00 29.56 A ATOM 977 CD1 PHE A 203 12.014 −9.630 64.460 1.0027.71 A ATOM 978 CD2 PHE A 203 12.609 −9.375 62.154 1.00 27.89 A ATOM979 CE1 PHE A 203 13.348 −9.884 64.789 1.00 27.78 A ATOM 980 CE2 PHE A203 13.947 −9.626 62.471 1.00 31.39 A ATOM 981 CZ PHE A 203 14.317−9.882 63.793 1.00 29.74 A ATOM 982 C PHE A 203 7.842 −9.963 63.270 1.0027.04 A ATOM 983 O PHE A 203 7.502 −9.297 64.248 1.00 26.24 A ATOM 984 NLEU A 204 6.975 −10.380 62.350 1.00 27.91 A ATOM 985 CA LEU A 204 5.551−10.078 62.479 1.00 26.99 A ATOM 986 CB LEU A 204 4.797 −10.467 61.2011.00 28.39 A ATOM 987 CG LEU A 204 4.955 −9.565 59.972 1.00 28.86 A ATOM988 CD1 LEU A 204 4.344 −10.225 58.766 1.00 30.50 A ATOM 989 CD2 LEU A204 4.289 −8.219 60.229 1.00 30.89 A ATOM 990 C LEU A 204 4.983 −10.86463.662 1.00 27.43 A ATOM 991 O LEU A 204 4.069 −10.402 64.341 1.00 27.90A ATOM 992 N VAL A 205 5.530 −12.055 63.908 1.00 26.39 A ATOM 993 CA VALA 205 5.052 −12.889 65.005 1.00 26.90 A ATOM 994 CB VAL A 205 4.616−14.283 64.497 1.00 28.55 A ATOM 995 CG1 VAL A 205 3.486 −14.143 63.4831.00 25.90 A ATOM 996 CG2 VAL A 205 5.814 −15.014 63.884 1.00 27.28 AATOM 997 C VAL A 205 6.059 −13.115 66.135 1.00 27.16 A ATOM 998 O VAL A205 5.712 −13.667 67.175 1.00 26.02 A ATOM 999 N ALA A 206 7.301 −12.68965.936 1.00 26.54 A ATOM 1000 CA ALA A 206 8.327 −12.913 66.942 1.0028.79 A ATOM 1001 CB ALA A 206 9.396 −13.874 66.384 1.00 27.34 A ATOM1002 C ALA A 206 9.003 −11.663 67.483 1.00 28.08 A ATOM 1003 O ALA A 2069.336 −10.744 66.732 1.00 30.28 A ATOM 1004 N ASP A 207 9.202 −11.64568.798 1.00 27.34 A ATOM 1005 CA ASP A 207 9.888 −10.543 69.459 1.0027.28 A ATOM 1006 CB ASP A 207 9.498 −10.497 70.935 1.00 29.37 A ATOM1007 CG ASP A 207 8.166 −9.819 71.154 1.00 29.88 A ATOM 1008 OD1 ASP A207 7.426 −10.251 72.060 1.00 33.62 A ATOM 1009 OD2 ASP A 207 7.872−8.851 70.415 1.00 28.84 A ATOM 1010 C ASP A 207 11.391 −10.781 69.3251.00 27.50 A ATOM 1011 O ASP A 207 12.191 −9.848 69.383 1.00 28.52 AATOM 1012 N LYS A 208 11.759 −12.045 69.130 1.00 24.98 A ATOM 1013 CALYS A 208 13.158 −12.434 68.976 1.00 29.19 A ATOM 1014 CB LYS A 20813.733 −12.875 70.326 1.00 30.51 A ATOM 1015 CG LYS A 208 15.046 −13.62770.216 1.00 34.73 A ATOM 1016 CD LYS A 208 16.243 −12.747 70.521 1.0037.07 A ATOM 1017 CE LYS A 208 16.357 −12.473 72.009 1.00 35.69 A ATOM1018 NZ LYS A 208 17.669 −11.850 72.336 1.00 38.56 A ATOM 1019 C LYS A208 13.312 −13.574 67.970 1.00 27.58 A ATOM 1020 O LYS A 208 12.540−14.526 67.979 1.00 26.32 A ATOM 1021 N VAL A 209 14.312 −13.465 67.1041.00 25.41 A ATOM 1022 CA VAL A 209 14.575 −14.501 66.114 1.00 25.21 AATOM 1023 CB VAL A 209 14.447 −13.963 64.665 1.00 24.58 A ATOM 1024 CG1VAL A 209 14.799 −15.070 63.660 1.00 24.08 A ATOM 1025 CG2 VAL A 20913.016 −13.471 64.419 1.00 24.90 A ATOM 1026 C VAL A 209 15.988 −15.02466.339 1.00 27.03 A ATOM 1027 O VAL A 209 16.948 −14.256 66.400 1.0025.92 A ATOM 1028 N ILE A 210 16.101 −16.337 66.480 1.00 24.68 A ATOM1029 CA ILE A 210 17.381 −16.966 66.694 1.00 26.11 A ATOM 1030 CB ILE A210 17.398 −17.700 68.048 1.00 27.89 A ATOM 1031 CG2 ILE A 210 18.797−18.221 68.337 1.00 26.45 A ATOM 1032 CG1 ILE A 210 16.940 −16.74069.155 1.00 28.54 A ATOM 1033 CD1 ILE A 210 16.938 −17.347 70.523 1.0032.12 A ATOM 1034 C ILE A 210 17.653 −17.960 65.563 1.00 26.52 A ATOM1035 O ILE A 210 16.774 −18.736 65.167 1.00 24.24 A ATOM 1036 N VAL A211 18.872 −17.925 65.037 1.00 25.00 A ATOM 1037 CA VAL A 211 19.247−18.825 63.961 1.00 25.48 A ATOM 1038 CB VAL A 211 19.536 −18.051 62.6411.00 26.62 A ATOM 1039 CG1 VAL A 211 19.905 −19.030 61.539 1.00 24.54 AATOM 1040 CG2 VAL A 211 18.309 −17.223 62.225 1.00 25.83 A ATOM 1041 CVAL A 211 20.488 −19.626 64.350 1.00 25.39 A ATOM 1042 O VAL A 21121.578 −19.075 64.492 1.00 25.48 A ATOM 1043 N THR A 212 20.312 −20.92664.551 1.00 25.49 A ATOM 1044 CA THR A 212 21.444 −21.777 64.883 1.0025.56 A ATOM 1045 CB THR A 212 21.089 −22.788 65.983 1.00 28.29 A ATOM1046 OG1 THR A 212 20.580 −22.082 67.123 1.00 26.38 A ATOM 1047 CG2 THRA 212 22.337 −23.577 66.401 1.00 26.88 A ATOM 1048 C THR A 212 21.778−22.487 63.579 1.00 25.09 A ATOM 1049 O THR A 212 20.896 −23.037 62.9211.00 23.68 A ATOM 1050 N SER A 213 23.045 −22.470 63.187 1.00 25.58 AATOM 1051 CA SER A 213 23.394 −23.079 61.916 1.00 27.51 A ATOM 1052 CBSER A 213 23.306 −22.005 60.819 1.00 24.34 A ATOM 1053 OG SER A 21323.316 −22.565 59.521 1.00 25.66 A ATOM 1054 C SER A 213 24.766 −23.73661.892 1.00 29.07 A ATOM 1055 O SER A 213 25.724 −23.223 62.473 1.0030.35 A ATOM 1056 N LYS A 214 24.838 −24.872 61.202 1.00 29.96 A ATOM1057 CA LYS A 214 26.072 −25.637 61.044 1.00 29.56 A ATOM 1058 CB LYS A214 26.002 −26.959 61.817 1.00 30.30 A ATOM 1059 CG LYS A 214 27.229−27.867 61.623 1.00 31.55 A ATOM 1060 CD LYS A 214 28.509 −27.189 62.1011.00 31.08 A ATOM 1061 CE LYS A 214 29.749 −28.087 61.948 1.00 32.51 AATOM 1062 NZ LYS A 214 30.169 −28.275 60.520 1.00 31.25 A ATOM 1063 CLYS A 214 26.281 −25.929 59.561 1.00 28.85 A ATOM 1064 O LYS A 21425.490 −26.632 58.933 1.00 28.51 A ATOM 1065 N HIS A 215 27.345 −25.35659.014 1.00 31.48 A ATOM 1066 CA HIS A 215 27.719 −25.525 57.614 1.0032.33 A ATOM 1067 CB HIS A 215 27.997 −24.150 57.005 1.00 32.68 A ATOM1068 CG HIS A 215 28.395 −24.185 55.564 1.00 35.07 A ATOM 1069 CD2 HIS A215 29.575 −24.486 54.971 1.00 35.69 A ATOM 1070 ND1 HIS A 215 27.526−23.855 54.546 1.00 37.05 A ATOM 1071 CE1 HIS A 215 28.154 −23.94953.386 1.00 36.12 A ATOM 1072 NE2 HIS A 215 29.398 −24.330 53.616 1.0038.37 A ATOM 1073 C HIS A 215 28.996 −26.377 57.606 1.00 33.45 A ATOM1074 O HIS A 215 29.836 −26.238 58.496 1.00 34.03 A ATOM 1075 N ASN A216 29.140 −27.249 56.609 1.00 34.16 A ATOM 1076 CA ASN A 216 30.313−28.117 56.505 1.00 34.54 A ATOM 1077 CB ASN A 216 30.309 −28.901 55.1801.00 33.51 A ATOM 1078 CG ASN A 216 29.247 −29.968 55.137 1.00 31.18 AATOM 1079 OD1 ASN A 216 28.825 −30.469 56.170 1.00 34.13 A ATOM 1080 ND2ASN A 216 28.816 −30.336 53.932 1.00 32.44 A ATOM 1081 C ASN A 21631.656 −27.408 56.617 1.00 34.35 A ATOM 1082 O ASN A 216 32.633 −28.00657.054 1.00 35.40 A ATOM 1083 N ASN A 217 31.713 −26.142 56.230 1.0034.75 A ATOM 1084 CA ASN A 217 32.975 −25.411 56.264 1.00 34.72 A ATOM1085 CB ASN A 217 33.090 −24.520 55.026 1.00 38.42 A ATOM 1086 CG ASN A217 33.241 −25.319 53.738 1.00 41.13 A ATOM 1087 OD1 ASN A 217 33.142−24.770 52.636 1.00 43.67 A ATOM 1088 ND2 ASN A 217 33.489 −26.62153.871 1.00 40.39 A ATOM 1089 C ASN A 217 33.200 −24.569 57.506 1.0035.80 A ATOM 1090 O ASN A 217 34.084 −23.714 57.523 1.00 36.93 A ATOM1091 N ASP A 218 32.415 −24.802 58.551 1.00 35.43 A ATOM 1092 CA ASP A218 32.575 −24.024 59.771 1.00 35.16 A ATOM 1093 CB ASP A 218 31.906−22.654 59.606 1.00 36.23 A ATOM 1094 CG ASP A 218 32.519 −21.595 60.4971.00 37.78 A ATOM 1095 OD1 ASP A 218 33.285 −21.958 61.412 1.00 38.97 AATOM 1096 OD2 ASP A 218 32.235 −20.398 60.286 1.00 37.63 A ATOM 1097 CASP A 218 31.963 −24.749 60.958 1.00 34.91 A ATOM 1098 O ASP A 21831.336 −25.794 60.802 1.00 36.71 A ATOM 1099 N THR A 219 32.158 −24.19862.149 1.00 34.34 A ATOM 1100 CA THR A 219 31.590 −24.791 63.350 1.0035.03 A ATOM 1101 CB THR A 219 32.438 −24.459 64.588 1.00 36.33 A ATOM1102 OG1 THR A 219 32.620 −23.041 64.671 1.00 37.48 A ATOM 1103 CG2 THRA 219 33.809 −25.140 64.493 1.00 36.36 A ATOM 1104 C THR A 219 30.190−24.207 63.517 1.00 34.77 A ATOM 1105 O THR A 219 29.787 −23.325 62.7531.00 34.10 A ATOM 1106 N GLN A 220 29.455 −24.689 64.513 1.00 33.81 AATOM 1107 CA GLN A 220 28.095 −24.220 64.749 1.00 33.03 A ATOM 1108 CBGLN A 220 27.346 −25.215 65.630 1.00 34.22 A ATOM 1109 CG GLN A 22025.861 −24.923 65.771 1.00 32.44 A ATOM 1110 CD GLN A 220 25.081 −26.15566.160 1.00 33.58 A ATOM 1111 OE1 GLN A 220 25.181 −27.195 65.505 1.0035.72 A ATOM 1112 NE2 GLN A 220 24.291 −26.050 67.222 1.00 32.58 A ATOM1113 C GLN A 220 28.042 −22.835 65.378 1.00 33.09 A ATOM 1114 O GLN A220 28.769 −22.539 66.326 1.00 33.09 A ATOM 1115 N HIS A 221 27.169−21.989 64.843 1.00 32.12 A ATOM 1116 CA HIS A 221 27.025 −20.632 65.3501.00 31.17 A ATOM 1117 CB HIS A 221 27.652 −19.636 64.368 1.00 31.00 AATOM 1118 CG HIS A 221 29.145 −19.735 64.281 1.00 34.24 A ATOM 1119 CD2HIS A 221 29.943 −20.545 63.548 1.00 32.61 A ATOM 1120 ND1 HIS A 22129.990 −18.953 65.041 1.00 36.07 A ATOM 1121 CE1 HIS A 221 31.244−19.277 64.778 1.00 34.47 A ATOM 1122 NE2 HIS A 221 31.243 −20.24063.876 1.00 35.07 A ATOM 1123 C HIS A 221 25.572 −20.263 65.608 1.0030.98 A ATOM 1124 O HIS A 221 24.650 −20.929 65.129 1.00 27.49 A ATOM1125 N ILE A 222 25.385 −19.202 66.386 1.00 28.25 A ATOM 1126 CA ILE A222 24.063 −18.713 66.729 1.00 28.60 A ATOM 1127 CB ILE A 222 23.772−18.904 68.227 1.00 28.80 A ATOM 1128 CG2 ILE A 222 22.354 −18.42968.543 1.00 27.64 A ATOM 1129 CG1 ILE A 222 23.946 −20.377 68.601 1.0027.19 A ATOM 1130 CD1 ILE A 222 23.890 −20.640 70.104 1.00 28.34 A ATOM1131 C ILE A 222 23.939 −17.229 66.405 1.00 27.81 A ATOM 1132 O ILE A222 24.779 −16.424 66.801 1.00 27.24 A ATOM 1133 N TRP A 223 22.893−16.892 65.656 1.00 28.88 A ATOM 1134 CA TRP A 223 22.590 −15.516 65.2691.00 26.45 A ATOM 1135 CB TRP A 223 22.313 −15.456 63.761 1.00 26.37 AATOM 1136 CG TRP A 223 21.935 −14.104 63.195 1.00 24.42 A ATOM 1137 CD2TRP A 223 20.611 −13.551 63.091 1.00 23.57 A ATOM 1138 CE2 TRP A 22320.722 −12.323 62.396 1.00 24.40 A ATOM 1139 CE3 TRP A 223 19.342−13.976 63.512 1.00 24.61 A ATOM 1140 CD1 TRP A 223 22.768 −13.20962.590 1.00 22.60 A ATOM 1141 NE1 TRP A 223 22.048 −12.139 62.103 1.0024.95 A ATOM 1142 CZ2 TRP A 223 19.611 −11.512 62.112 1.00 22.27 A ATOM1143 CZ3 TRP A 223 18.233 −13.168 63.226 1.00 23.64 A ATOM 1144 CH2 TRPA 223 18.380 −11.952 62.532 1.00 21.24 A ATOM 1145 C TRP A 223 21.320−15.211 66.055 1.00 26.45 A ATOM 1146 O TRP A 223 20.431 −16.054 66.1451.00 25.73 A ATOM 1147 N GLU A 224 21.237 −14.028 66.645 1.00 27.69 AATOM 1148 CA GLU A 224 20.047 −13.670 67.410 1.00 28.57 A ATOM 1149 CBGLU A 224 20.236 −14.049 68.882 1.00 28.25 A ATOM 1150 CG GLU A 22421.394 −13.352 69.561 1.00 32.38 A ATOM 1151 CD GLU A 224 20.996 −12.04070.203 1.00 35.31 A ATOM 1152 OE1 GLU A 224 21.908 −11.266 70.560 1.0037.89 A ATOM 1153 OE2 GLU A 224 19.779 −11.787 70.362 1.00 34.10 A ATOM1154 C GLU A 224 19.755 −12.181 67.254 1.00 26.84 A ATOM 1155 O GLU A224 20.670 −11.365 67.201 1.00 26.00 A ATOM 1156 N SER A 225 18.476−11.832 67.179 1.00 26.97 A ATOM 1157 CA SER A 225 18.089 −10.436 66.9851.00 27.37 A ATOM 1158 CB SER A 225 18.284 −10.084 65.504 1.00 27.31 AATOM 1159 OG SER A 225 17.842 −8.778 65.204 1.00 27.10 A ATOM 1160 C SERA 225 16.643 −10.136 67.383 1.00 27.99 A ATOM 1161 O SER A 225 15.772−10.997 67.291 1.00 27.02 A ATOM 1162 N ASP A 226 16.401 −8.908 67.8301.00 29.13 A ATOM 1163 CA ASP A 226 15.054 −8.466 68.185 1.00 28.16 AATOM 1164 CB ASP A 226 15.027 −7.811 69.568 1.00 28.71 A ATOM 1165 CGASP A 226 15.886 −6.565 69.641 1.00 29.58 A ATOM 1166 OD1 ASP A 22615.726 −5.803 70.610 1.00 33.74 A ATOM 1167 OD2 ASP A 226 16.727 −6.34668.742 1.00 30.11 A ATOM 1168 C ASP A 226 14.692 −7.434 67.131 1.0028.91 A ATOM 1169 O ASP A 226 13.724 −6.689 67.276 1.00 29.07 A ATOM1170 N SER A 227 15.515 −7.406 66.082 1.00 26.17 A ATOM 1171 CA SER A227 15.399 −6.510 64.930 1.00 27.87 A ATOM 1172 CB SER A 227 13.933−6.314 64.516 1.00 26.81 A ATOM 1173 OG SER A 227 13.322 −5.256 65.2341.00 30.30 A ATOM 1174 C SER A 227 16.059 −5.137 65.132 1.00 25.84 AATOM 1175 O SER A 227 16.197 −4.371 64.182 1.00 25.78 A ATOM 1176 N ASNA 228 16.474 −4.834 66.357 1.00 25.68 A ATOM 1177 CA ASN A 228 17.106−3.544 66.654 1.00 26.22 A ATOM 1178 CB ASN A 228 16.692 −3.079 68.0481.00 26.07 A ATOM 1179 CG ASN A 228 15.209 −2.802 68.142 1.00 30.49 AATOM 1180 OD1 ASN A 228 14.535 −3.270 69.061 1.00 34.03 A ATOM 1181 ND2ASN A 228 14.692 −2.034 67.191 1.00 22.67 A ATOM 1182 C ASN A 228 18.628−3.615 66.567 1.00 26.12 A ATOM 1183 O ASN A 228 19.322 −2.605 66.7131.00 25.83 A ATOM 1184 N ALA A 229 19.119 −4.827 66.333 1.00 24.72 AATOM 1185 CA ALA A 229 20.538 −5.140 66.203 1.00 25.83 A ATOM 1186 CBALA A 229 21.324 −4.626 67.425 1.00 24.69 A ATOM 1187 C ALA A 229 20.568−6.661 66.173 1.00 26.02 A ATOM 1188 O ALA A 229 19.521 −7.300 66.2871.00 24.68 A ATOM 1189 N PHE A 230 21.753 −7.240 66.013 1.00 25.02 AATOM 1190 CA PHE A 230 21.883 −8.689 66.023 1.00 28.61 A ATOM 1191 CBPHE A 230 21.562 −9.285 64.639 1.00 27.93 A ATOM 1192 CG PHE A 23022.631 −9.067 63.595 1.00 26.10 A ATOM 1193 CD1 PHE A 230 23.700 −9.95363.478 1.00 25.19 A ATOM 1194 CD2 PHE A 230 22.537 −8.007 62.693 1.0024.79 A ATOM 1195 CE1 PHE A 230 24.653 −9.793 62.478 1.00 23.58 A ATOM1196 CE2 PHE A 230 23.488 −7.836 61.688 1.00 24.57 A ATOM 1197 CZ PHE A230 24.550 −8.736 61.579 1.00 24.47 A ATOM 1198 C PHE A 230 23.287−9.061 66.459 1.00 28.38 A ATOM 1199 O PHE A 230 24.203 −8.254 66.3611.00 30.46 A ATOM 1200 N SER A 231 23.451 −10.272 66.974 1.00 30.93 AATOM 1201 CA SER A 231 24.768 −10.725 67.396 1.00 31.70 A ATOM 1202 CBSER A 231 24.914 −10.627 68.916 1.00 30.54 A ATOM 1203 OG SER A 23124.132 −11.611 69.558 1.00 34.51 A ATOM 1204 C SER A 231 24.996 −12.16166.949 1.00 30.94 A ATOM 1205 O SER A 231 24.051 −12.893 66.645 1.0028.43 A ATOM 1206 N VAL A 232 26.262 −12.553 66.897 1.00 31.28 A ATOM1207 CA VAL A 232 26.628 −13.902 66.500 1.00 34.01 A ATOM 1208 CB VAL A232 27.173 −13.944 65.053 1.00 34.38 A ATOM 1209 CG1 VAL A 232 28.317−12.965 64.899 1.00 38.67 A ATOM 1210 CG2 VAL A 232 27.648 −15.35064.718 1.00 35.03 A ATOM 1211 C VAL A 232 27.696 −14.438 67.436 1.0033.64 A ATOM 1212 O VAL A 232 28.686 −13.768 67.722 1.00 34.60 A ATOM1213 N ILE A 233 27.482 −15.651 67.919 1.00 34.01 A ATOM 1214 CA ILE A233 28.429 −16.282 68.816 1.00 32.94 A ATOM 1215 CB ILE A 233 27.894−16.340 70.256 1.00 34.86 A ATOM 1216 CG2 ILE A 233 27.550 −14.94370.750 1.00 36.09 A ATOM 1217 CG1 ILE A 233 26.662 −17.251 70.307 1.0033.82 A ATOM 1218 CD1 ILE A 233 26.219 −17.606 71.713 1.00 35.91 A ATOM1219 C ILE A 233 28.638 −17.717 68.367 1.00 32.97 A ATOM 1220 O ILE A233 27.757 −18.310 67.747 1.00 30.39 A ATOM 1221 N ALA A 234 29.806−18.270 68.670 1.00 32.69 A ATOM 1222 CA ALA A 234 30.061 −19.660 68.3381.00 33.35 A ATOM 1223 CB ALA A 234 31.508 −20.023 68.649 1.00 31.89 AATOM 1224 C ALA A 234 29.115 −20.373 69.297 1.00 32.93 A ATOM 1225 O ALAA 234 28.931 −19.920 70.426 1.00 33.49 A ATOM 1226 N ASP A 235 28.495−21.458 68.852 1.00 33.92 A ATOM 1227 CA ASP A 235 27.565 −22.195 69.7031.00 35.07 A ATOM 1228 CB ASP A 235 26.740 −23.172 68.862 1.00 32.85 AATOM 1229 CG ASP A 235 25.509 −23.673 69.589 1.00 34.03 A ATOM 1230 OD1ASP A 235 25.567 −23.815 70.824 1.00 35.68 A ATOM 1231 OD2 ASP A 23524.483 −23.938 68.926 1.00 34.84 A ATOM 1232 C ASP A 235 28.345 −22.96670.772 1.00 37.82 A ATOM 1233 O ASP A 235 29.114 −23.878 70.457 1.0036.64 A ATOM 1234 N PRO A 236 28.154 −22.612 72.056 1.00 40.23 A ATOM1235 CD PRO A 236 27.189 −21.645 72.613 1.00 40.64 A ATOM 1236 CA PRO A236 28.872 −23.310 73.129 1.00 40.88 A ATOM 1237 CB PRO A 236 28.469−22.524 74.378 1.00 41.33 A ATOM 1238 CG PRO A 236 27.065 −22.102 74.0531.00 41.51 A ATOM 1239 C PRO A 236 28.500 −24.789 73.223 1.00 41.29 AATOM 1240 O PRO A 236 29.231 −25.581 73.816 1.00 41.99 A ATOM 1241 N ARGA 237 27.368 −25.157 72.628 1.00 40.37 A ATOM 1242 CA ARG A 237 26.900−26.542 72.653 1.00 39.76 A ATOM 1243 CB ARG A 237 25.396 −26.597 72.3731.00 39.62 A ATOM 1244 CG ARG A 237 24.532 −25.892 73.393 1.00 38.78 AATOM 1245 CD ARG A 237 23.089 −25.803 72.912 1.00 39.41 A ATOM 1246 NEARG A 237 22.978 −24.977 71.713 1.00 35.90 A ATOM 1247 CZ ARG A 23721.829 −24.689 71.107 1.00 39.52 A ATOM 1248 NH1 ARG A 237 20.679−25.162 71.589 1.00 34.04 A ATOM 1249 NH2 ARG A 237 21.827 −23.92770.018 1.00 37.40 A ATOM 1250 C ARG A 237 27.617 −27.417 71.624 1.0041.21 A ATOM 1251 O ARG A 237 27.326 −28.610 71.506 1.00 40.45 A ATOM1252 N GLY A 238 28.545 −26.821 70.881 1.00 41.59 A ATOM 1253 CA GLY A238 29.261 −27.563 69.860 1.00 42.55 A ATOM 1254 C GLY A 238 28.378−27.872 68.660 1.00 43.55 A ATOM 1255 O GLY A 238 27.328 −27.250 68.4631.00 41.70 A ATOM 1256 N ASN A 239 28.801 −28.837 67.852 1.00 44.11 AATOM 1257 CA ASN A 239 28.041 −29.231 66.672 1.00 44.67 A ATOM 1258 CBASN A 239 28.978 −29.857 65.632 1.00 46.49 A ATOM 1259 CG ASN A 23928.238 −30.685 64.597 1.00 48.61 A ATOM 1260 OD1 ASN A 239 27.163−30.304 64.128 1.00 50.81 A ATOM 1261 ND2 ASN A 239 28.817 −31.82064.225 1.00 50.33 A ATOM 1262 C ASN A 239 26.928 −30.211 67.026 1.0043.82 A ATOM 1263 O ASN A 239 27.159 −31.415 67.095 1.00 46.79 A ATOM1264 N THR A 240 25.720 −29.695 67.244 1.00 40.25 A ATOM 1265 CA THR A240 24.584 −30.546 67.588 1.00 37.66 A ATOM 1266 CB THR A 240 23.759−29.958 68.756 1.00 38.88 A ATOM 1267 OG1 THR A 240 23.201 −28.69968.357 1.00 37.45 A ATOM 1268 CG2 THR A 240 24.632 −29.763 69.990 1.0035.95 A ATOM 1269 C THR A 240 23.635 −30.751 66.416 1.00 36.12 A ATOM1270 O THR A 240 22.773 −31.630 66.455 1.00 35.77 A ATOM 1271 N LEU A241 23.777 −29.933 65.380 1.00 35.46 A ATOM 1272 CA LEU A 241 22.915−30.057 64.211 1.00 34.67 A ATOM 1273 CB LEU A 241 22.696 −28.680 63.5601.00 32.39 A ATOM 1274 CG LEU A 241 21.837 −27.691 64.357 1.00 32.41 AATOM 1275 CD1 LEU A 241 21.794 −26.340 63.664 1.00 30.77 A ATOM 1276 CD2LEU A 241 20.443 −28.254 64.513 1.00 31.49 A ATOM 1277 C LEU A 24123.467 −31.037 63.174 1.00 32.75 A ATOM 1278 O LEU A 241 22.707 −31.62862.414 1.00 32.61 A ATOM 1279 N GLY A 242 24.781 −31.225 63.160 1.0032.82 A ATOM 1280 CA GLY A 242 25.384 −32.113 62.176 1.00 33.49 A ATOM1281 C GLY A 242 25.607 −31.244 60.954 1.00 32.75 A ATOM 1282 O GLY A242 26.743 −30.972 60.560 1.00 33.31 A ATOM 1283 N ARG A 243 24.494−30.800 60.374 1.00 32.49 A ATOM 1284 CA ARG A 243 24.473 −29.903 59.2261.00 31.35 A ATOM 1285 CB ARG A 243 24.797 −30.621 57.912 1.00 31.73 AATOM 1286 CG ARG A 243 24.934 −29.641 56.738 1.00 32.57 A ATOM 1287 CDARG A 243 24.839 −30.310 55.370 1.00 30.58 A ATOM 1288 NE ARG A 24323.582 −31.031 55.212 1.00 30.37 A ATOM 1289 CZ ARG A 243 23.442 −32.33855.409 1.00 28.39 A ATOM 1290 NH1 ARG A 243 24.489 −33.077 55.763 1.0029.52 A ATOM 1291 NH2 ARG A 243 22.254 −32.900 55.262 1.00 27.01 A ATOM1292 C ARG A 243 23.056 −29.347 59.131 1.00 31.98 A ATOM 1293 O ARG A243 22.081 −30.075 59.314 1.00 29.48 A ATOM 1294 N GLY A 244 22.936−28.057 58.848 1.00 31.79 A ATOM 1295 CA GLY A 244 21.611 −27.490 58.7351.00 31.73 A ATOM 1296 C GLY A 244 21.385 −26.208 59.501 1.00 28.94 AATOM 1297 O GLY A 244 22.325 −25.545 59.938 1.00 28.30 A ATOM 1298 N THRA 245 20.112 −25.882 59.693 1.00 28.92 A ATOM 1299 CA THR A 245 19.748−24.646 60.359 1.00 27.12 A ATOM 1300 CB THR A 245 19.585 −23.514 59.3171.00 29.13 A ATOM 1301 OG1 THR A 245 20.752 −23.457 58.484 1.00 28.18 AATOM 1302 CG2 THR A 245 19.374 −22.161 60.008 1.00 29.18 A ATOM 1303 CTHR A 245 18.448 −24.751 61.129 1.00 28.44 A ATOM 1304 O THR A 24517.491 −25.396 60.683 1.00 29.55 A ATOM 1305 N THR A 246 18.421 −24.11662.294 1.00 27.00 A ATOM 1306 CA THR A 246 17.220 −24.080 63.104 1.0027.61 A ATOM 1307 CB THR A 246 17.414 −24.659 64.529 1.00 29.89 A ATOM1308 OG1 THR A 246 17.545 −26.084 64.469 1.00 30.34 A ATOM 1309 CG2 THRA 246 16.201 −24.307 65.407 1.00 30.40 A ATOM 1310 C THR A 246 16.834−22.621 63.276 1.00 26.97 A ATOM 1311 O THR A 246 17.636 −21.802 63.7411.00 27.17 A ATOM 1312 N ILE A 247 15.615 −22.294 62.880 1.00 25.19 AATOM 1313 CA ILE A 247 15.124 −20.944 63.054 1.00 25.58 A ATOM 1314 CBILE A 247 14.233 −20.479 61.887 1.00 25.50 A ATOM 1315 CG2 ILE A 24713.833 −19.021 62.114 1.00 25.21 A ATOM 1316 CG1 ILE A 247 14.947−20.684 60.541 1.00 24.91 A ATOM 1317 CD1 ILE A 247 16.199 −19.85460.345 1.00 24.45 A ATOM 1318 C ILE A 247 14.247 −21.081 64.285 1.0024.63 A ATOM 1319 O ILE A 247 13.279 −21.838 64.276 1.00 25.24 A ATOM1320 N THR A 248 14.603 −20.377 65.349 1.00 25.95 A ATOM 1321 CA THR A248 13.830 −20.424 66.584 1.00 25.55 A ATOM 1322 CB THR A 248 14.738−20.727 67.801 1.00 26.69 A ATOM 1323 OG1 THR A 248 15.296 −22.04367.673 1.00 28.48 A ATOM 1324 CG2 THR A 248 13.933 −20.640 69.104 1.0026.02 A ATOM 1325 C THR A 248 13.153 −19.075 66.784 1.00 26.59 A ATOM1326 O THR A 248 13.813 −18.036 66.806 1.00 26.71 A ATOM 1327 N LEU A249 11.835 −19.089 66.920 1.00 26.73 A ATOM 1328 CA LEU A 249 11.103−17.851 67.122 1.00 28.46 A ATOM 1329 CB LEU A 249 9.958 −17.737 66.1091.00 26.10 A ATOM 1330 CG LEU A 249 10.287 −17.887 64.618 1.00 27.20 AATOM 1331 CD1 LEU A 249 9.060 −17.498 63.811 1.00 28.67 A ATOM 1332 CD2LEU A 249 11.458 −17.012 64.227 1.00 25.85 A ATOM 1333 C LEU A 24910.535 −17.745 68.534 1.00 28.32 A ATOM 1334 O LEU A 249 9.859 −18.65569.014 1.00 28.30 A ATOM 1335 N VAL A 250 10.846 −16.643 69.208 1.0028.75 A ATOM 1336 CA VAL A 250 10.318 −16.388 70.542 1.00 29.14 A ATOM1337 CB VAL A 250 11.298 −15.549 71.387 1.00 29.73 A ATOM 1338 CG1 VAL A250 10.701 −15.274 72.763 1.00 29.16 A ATOM 1339 CG2 VAL A 250 12.628−16.282 71.515 1.00 27.56 A ATOM 1340 C VAL A 250 9.057 −15.580 70.2341.00 30.79 A ATOM 1341 O VAL A 250 9.114 −14.369 70.015 1.00 30.24 AATOM 1342 N LEU A 251 7.924 −16.274 70.208 1.00 31.70 A ATOM 1343 CA LEUA 251 6.639 −15.684 69.857 1.00 33.83 A ATOM 1344 CB LEU A 251 5.596−16.803 69.749 1.00 33.73 A ATOM 1345 CG LEU A 251 5.914 −17.822 68.6491.00 35.88 A ATOM 1346 CD1 LEU A 251 4.901 −18.952 68.679 1.00 34.78 AATOM 1347 CD2 LEU A 251 5.916 −17.121 67.288 1.00 39.61 A ATOM 1348 CLEU A 251 6.066 −14.539 70.693 1.00 34.58 A ATOM 1349 O LEU A 251 6.113−14.552 71.922 1.00 31.56 A ATOM 1350 N LYS A 252 5.522 −13.547 69.9951.00 35.31 A ATOM 1351 CA LYS A 252 4.894 −12.414 70.652 1.00 37.92 AATOM 1352 CB LYS A 252 4.429 −11.371 69.633 1.00 36.52 A ATOM 1353 CGLYS A 252 5.531 −10.673 68.857 1.00 37.81 A ATOM 1354 CD LYS A 252 4.929−9.718 67.829 1.00 35.73 A ATOM 1355 ICE LYS A 252 6.004 −8.907 67.1291.00 35.52 A ATOM 1356 NZ LYS A 252 6.722 −8.038 68.096 1.00 34.94 AATOM 1357 C LYS A 252 3.668 −12.975 71.353 1.00 40.30 A ATOM 1358 O LYSA 252 3.095 −13.980 70.920 1.00 36.25 A ATOM 1359 N GLU A 253 3.263−12.319 72.431 1.00 43.28 A ATOM 1360 CA GLU A 253 2.095 −12.746 73.1881.00 46.90 A ATOM 1361 CB GLU A 253 1.740 −11.669 74.215 1.00 50.77 AATOM 1362 CG GLU A 253 0.690 −12.076 75.232 1.00 57.75 A ATOM 1363 CDGLU A 253 0.475 −11.008 76.291 1.00 60.96 A ATOM 1364 OE1 GLU A 2531.456 −10.636 76.974 1.00 61.81 A ATOM 1365 OE2 GLU A 253 −0.674 −10.54076.438 1.00 63.03 A ATOM 1366 C GLU A 253 0.914 −12.980 72.245 1.0045.72 A ATOM 1367 O GLU A 253 0.253 −14.015 72.303 1.00 45.27 A ATOM1368 N GLU A 254 0.679 −12.018 71.360 1.00 45.23 A ATOM 1369 CA GLU A254 −0.422 −12.077 70.405 1.00 47.70 A ATOM 1370 CB GLU A 254 −0.560−10.729 69.686 1.00 50.07 A ATOM 1371 CG GLU A 254 −0.180 −9.528 70.5401.00 54.85 A ATOM 1372 CD GLU A 254 1.324 −9.360 70.667 1.00 56.21 AATOM 1373 OE1 GLU A 254 1.951 −8.923 69.680 1.00 56.48 A ATOM 1374 OE2GLU A 254 1.879 −9.673 71.744 1.00 58.20 A ATOM 1375 C GLU A 254 −0.299−13.178 69.356 1.00 46.43 A ATOM 1376 O GLU A 254 −1.191 −13.334 68.5211.00 47.19 A ATOM 1377 N ALA A 255 0.790 −13.942 69.392 1.00 44.24 AATOM 1378 CA ALA A 255 0.995 −15.002 68.405 1.00 40.23 A ATOM 1379 CBALA A 255 2.275 −14.724 67.618 1.00 39.06 A ATOM 1380 C ALA A 255 1.043−16.408 68.998 1.00 39.13 A ATOM 1381 O ALA A 255 1.506 −17.347 68.3411.00 36.42 A ATOM 1382 N SER A 256 0.559 −16.559 70.229 1.00 39.57 AATOM 1383 CA SER A 256 0.566 −17.861 70.899 1.00 40.85 A ATOM 1384 CBSER A 256 −0.085 −17.759 72.287 1.00 42.00 A ATOM 1385 OG SER A 256−1.441 −17.356 72.201 1.00 46.83 A ATOM 1386 C SER A 256 −0.114 −18.96770.093 1.00 40.89 A ATOM 1387 O SER A 256 0.205 −20.143 70.257 1.0040.95 A ATOM 1388 N ASP A 257 −1.049 −18.592 69.227 1.00 41.73 A ATOM1389 CA ASP A 257 −1.747 −19.562 68.387 1.00 43.77 A ATOM 1390 CB ASP A257 −2.666 −18.845 67.400 1.00 47.93 A ATOM 1391 CG ASP A 257 −4.125−19.113 67.670 1.00 52.41 A ATOM 1392 OD1 ASP A 257 −4.491 −20.30467.800 1.00 54.34 A ATOM 1393 OD2 ASP A 257 −4.901 −18.134 67.744 1.0053.33 A ATOM 1394 C ASP A 257 −0.773 −20.431 67.592 1.00 44.09 A ATOM1395 O ASP A 257 −1.062 −21.592 67.294 1.00 43.05 A ATOM 1396 N TYR A258 0.375 −19.860 67.239 1.00 42.61 A ATOM 1397 CA TYR A 258 1.371−20.593 66.473 1.00 43.55 A ATOM 1398 CB TYR A 258 2.368 −19.621 65.8371.00 40.49 A ATOM 1399 CG TYR A 258 1.725 −18.682 64.840 1.00 37.93 AATOM 1400 CD1 TYR A 258 1.442 −17.360 65.175 1.00 37.43 A ATOM 1401 CE1TYR A 258 0.808 −16.507 64.271 1.00 38.22 A ATOM 1402 CD2 TYR A 2581.359 −19.133 63.572 1.00 38.12 A ATOM 1403 CE2 TYR A 258 0.725 −18.29362.660 1.00 39.12 A ATOM 1404 CZ TYR A 258 0.451 −16.982 63.014 1.0040.73 A ATOM 1405 OH TYR A 258 −0.186 −16.154 62.111 1.00 43.50 A ATOM1406 C TYR A 258 2.093 −21.620 67.336 1.00 44.26 A ATOM 1407 O TYR A 2583.070 −22.231 66.910 1.00 45.55 A ATOM 1408 N LEU A 259 1.605 −21.80068.558 1.00 45.18 A ATOM 1409 CA LEU A 259 2.178 −22.778 69.470 1.0045.20 A ATOM 1410 CB LEU A 259 2.267 −22.207 70.889 1.00 43.71 A ATOM1411 CG LEU A 259 3.271 −21.077 71.135 1.00 46.13 A ATOM 1412 CD1 LEU A259 3.118 −20.553 72.556 1.00 43.85 A ATOM 1413 CD2 LEU A 259 4.688−21.589 70.904 1.00 43.81 A ATOM 1414 C LEU A 259 1.279 −24.013 69.4611.00 45.63 A ATOM 1415 O LEU A 259 1.681 −25.085 69.911 1.00 44.85 AATOM 1416 N GLU A 260 0.064 −23.852 68.938 1.00 46.11 A ATOM 1417 CA GLUA 260 −0.905 −24.945 68.867 1.00 47.29 A ATOM 1418 CB GLU A 260 −2.329−24.389 68.752 1.00 48.35 A ATOM 1419 CG GLU A 260 −2.821 −23.653 69.9921.00 50.10 A ATOM 1420 CD GLU A 260 −2.796 −24.524 71.240 1.00 50.21 AATOM 1421 OE1 GLU A 260 −3.467 −25.576 71.260 1.00 52.40 A ATOM 1422 OE2GLU A 260 −2.103 −24.155 72.207 1.00 51.35 A ATOM 1423 C GLU A 260−0.629 −25.890 67.695 1.00 48.12 A ATOM 1424 O GLU A 260 −0.473 −25.45466.552 1.00 47.97 A ATOM 1425 N LEU A 261 −0.591 −27.186 67.994 1.0048.15 A ATOM 1426 CA LEU A 261 −0.318 −28.219 67.000 1.00 49.42 A ATOM1427 CB LEU A 261 −0.326 −29.604 67.657 1.00 50.55 A ATOM 1428 CG LEU A261 0.737 −29.856 68.726 1.00 52.10 A ATOM 1429 CD1 LEU A 261 0.611−31.278 69.246 1.00 52.02 A ATOM 1430 CD2 LEU A 261 2.119 −29.621 68.1361.00 53.44 A ATOM 1431 C LEU A 261 −1.267 −28.230 65.812 1.00 48.59 AATOM 1432 O LEU A 261 −0.834 −28.409 64.673 1.00 48.28 A ATOM 1433 N ASPA 262 −2.557 −28.055 66.064 1.00 47.88 A ATOM 1434 CA ASP A 262 −3.513−28.066 64.965 1.00 48.90 A ATOM 1435 CB ASP A 262 −4.950 −28.077 65.5001.00 51.04 A ATOM 1436 CG ASP A 262 −5.233 −26.930 66.441 1.00 54.48 AATOM 1437 OD1 ASP A 262 −4.535 −26.817 67.475 1.00 55.33 A ATOM 1438 OD2ASP A 262 −6.157 −26.143 66.148 1.00 57.16 A ATOM 1439 C ASP A 262−3.284 −26.869 64.046 1.00 48.05 A ATOM 1440 O ASP A 262 −3.399 −26.98262.823 1.00 46.94 A ATOM 1441 N THR A 263 −2.938 −25.727 64.632 1.0046.96 A ATOM 1442 CA THR A 263 −2.683 −24.531 63.837 1.00 46.87 A ATOM1443 CB THR A 263 −2.473 −23.291 64.731 1.00 47.54 A ATOM 1444 OG1 THR A263 −3.632 −23.089 65.551 1.00 49.30 A ATOM 1445 CG2 THR A 263 −2.240−22.047 63.872 1.00 45.04 A ATOM 1446 C THR A 263 −1.437 −24.718 62.9721.00 45.88 A ATOM 1447 O THR A 263 −1.478 −24.523 61.758 1.00 44.59 AATOM 1448 N ILE A 264 −0.334 −25.109 63.604 1.00 45.39 A ATOM 1449 CAILE A 264 0.921 −25.302 62.890 1.00 45.12 A ATOM 1450 CB ILE A 264 2.080−25.670 63.867 1.00 45.80 A ATOM 1451 CG2 ILE A 264 1.827 −27.018 64.5231.00 45.69 A ATOM 1452 CG1 ILE A 264 3.409 −25.705 63.111 1.00 45.29 AATOM 1453 CD1 ILE A 264 3.836 −24.354 62.576 1.00 48.05 A ATOM 1454 CILE A 264 0.820 −26.359 61.789 1.00 45.27 A ATOM 1455 O ILE A 264 1.221−26.107 60.652 1.00 43.64 A ATOM 1456 N LYS A 265 0.279 −27.532 62.1131.00 45.81 A ATOM 1457 CA LYS A 265 0.157 −28.595 61.115 1.00 46.73 AATOM 1458 CB LYS A 265 −0.501 −29.838 61.721 1.00 47.68 A ATOM 1459 CGLYS A 265 0.400 −30.575 62.703 1.00 50.09 A ATOM 1460 CD LYS A 265−0.121 −31.964 63.043 1.00 53.24 A ATOM 1461 CE LYS A 265 −1.399 −31.91563.861 1.00 55.91 A ATOM 1462 NZ LYS A 265 −1.849 −33.281 64.256 1.0060.96 A ATOM 1463 C LYS A 265 −0.622 −28.142 59.888 1.00 46.00 A ATOM1464 O LYS A 265 −0.225 −28.420 58.758 1.00 46.17 A ATOM 1465 N ASN A266 −1.721 −27.432 60.109 1.00 46.21 A ATOM 1466 CA ASN A 266 −2.540−26.948 59.007 1.00 47.31 A ATOM 1467 CB ASN A 266 −3.770 −26.216 59.5471.00 51.04 A ATOM 1468 CG ASN A 266 −4.830 −25.993 58.484 1.00 56.45 AATOM 1469 OD1 ASN A 266 −5.543 −26.922 58.094 1.00 59.75 A ATOM 1470 ND2ASN A 266 −4.934 −24.758 58.002 1.00 58.02 A ATOM 1471 C ASN A 266−1.720 −26.001 58.132 1.00 46.83 A ATOM 1472 O ASN A 266 −1.745 −26.09656.904 1.00 46.99 A ATOM 1473 N LEU A 267 −0.993 −25.087 58.770 1.0045.41 A ATOM 1474 CA LEU A 267 −0.166 −24.124 58.045 1.00 44.41 A ATOM1475 CB LEU A 267 0.408 −23.078 59.005 1.00 44.81 A ATOM 1476 CG LEU A267 −0.557 −22.100 59.675 1.00 44.89 A ATOM 1477 CD1 LEU A 267 0.220−21.201 60.626 1.00 45.00 A ATOM 1478 CD2 LEU A 267 −1.275 −21.26958.618 1.00 44.38 A ATOM 1479 C LEU A 267 0.981 −24.797 57.302 1.0042.78 A ATOM 1480 O LEU A 267 1.238 −24.490 56.139 1.00 42.03 A ATOM1481 N VAL A 268 1.676 −25.703 57.983 1.00 41.81 A ATOM 1482 CA VAL A268 2.798 −26.418 57.383 1.00 42.20 A ATOM 1483 CB VAL A 268 3.442−27.400 58.385 1.00 41.34 A ATOM 1484 CG1 VAL A 268 4.397 −28.338 57.6581.00 40.94 A ATOM 1485 CG2 VAL A 268 4.188 −26.628 59.460 1.00 39.92 AATOM 1486 C VAL A 268 2.348 −27.204 56.159 1.00 43.14 A ATOM 1487 O VALA 268 3.004 −27.187 55.119 1.00 41.73 A ATOM 1488 N LYS A 269 1.220−27.890 56.291 1.00 45.44 A ATOM 1489 CA LYS A 269 0.686 −28.687 55.1981.00 48.18 A ATOM 1490 CB LYS A 269 −0.511 −29.505 55.689 1.00 50.56 AATOM 1491 CG LYS A 269 −0.984 −30.560 54.707 1.00 55.15 A ATOM 1492 CDLYS A 269 −2.110 −31.396 55.305 1.00 58.30 A ATOM 1493 CE LYS A 269−2.590 −32.470 54.338 1.00 58.74 A ATOM 1494 NZ LYS A 269 −3.722 −33.25954.907 1.00 61.58 A ATOM 1495 C LYS A 269 0.273 −27.803 54.023 1.0047.87 A ATOM 1496 O LYS A 269 0.407 −28.196 52.865 1.00 48.99 A ATOM1497 N ALA A 270 −0.216 −26.605 54.322 1.00 47.71 A ATOM 1498 CA ALA A270 −0.647 −25.682 53.278 1.00 47.22 A ATOM 1499 CB ALA A 270 −1.518−24.593 53.880 1.00 47.69 A ATOM 1500 C ALA A 270 0.532 −25.049 52.5471.00 47.81 A ATOM 1501 O ALA A 270 0.407 −24.625 51.398 1.00 47.91 AATOM 1502 N TYR A 271 1.681 −24.998 53.210 1.00 46.43 A ATOM 1503 CA TYRA 271 2.867 −24.384 52.624 1.00 46.82 A ATOM 1504 CB TYR A 271 3.422−23.340 53.600 1.00 45.84 A ATOM 1505 CG TYR A 271 2.527 −22.136 53.8171.00 44.83 A ATOM 1506 CD1 TYR A 271 2.503 −21.477 55.049 1.00 43.90 AATOM 1507 CE1 TYR A 271 1.727 −20.341 55.244 1.00 44.66 A ATOM 1508 CD2TYR A 271 1.742 −21.624 52.782 1.00 43.86 A ATOM 1509 CE2 TYR A 2710.960 −20.485 52.965 1.00 44.32 A ATOM 1510 CZ TYR A 271 0.958 −19.84854.200 1.00 46.19 A ATOM 1511 OH TYR A 271 0.192 −18.715 54.392 1.0049.38 A ATOM 1512 C TYR A 271 3.982 −25.364 52.251 1.00 46.30 A ATOM1513 O TYR A 271 5.105 −24.944 51.971 1.00 45.31 A ATOM 1514 N SER A 2723.682 −26.659 52.228 1.00 46.53 A ATOM 1515 CA SER A 272 4.714 −27.64251.920 1.00 48.82 A ATOM 1516 CB SER A 272 5.038 −28.457 53.175 1.0049.85 A ATOM 1517 OG SER A 272 3.923 −29.228 53.591 1.00 51.28 A ATOM1518 C SER A 272 4.417 −28.601 50.773 1.00 49.33 A ATOM 1519 O SER A 2725.227 −29.481 50.477 1.00 48.33 A ATOM 1520 N ALA A 273 3.270 −28.43850.124 1.00 51.93 A ATOM 1521 CA ALA A 273 2.907 −29.320 49.019 1.0054.85 A ATOM 1522 CB ALA A 273 1.490 −29.002 48.543 1.00 54.46 A ATOM1523 C ALA A 273 3.891 −29.221 47.854 1.00 56.23 A ATOM 1524 O ALA A 2733.863 −30.041 46.937 1.00 57.06 A ATOM 1525 N PHE A 274 4.765 −28.21947.896 1.00 58.75 A ATOM 1526 CA PHE A 274 5.752 −28.010 46.836 1.0060.64 A ATOM 1527 CB PHE A 274 5.776 −26.536 46.421 1.00 63.61 A ATOM1528 CG PHE A 274 4.505 −26.058 45.773 1.00 66.68 A ATOM 1529 CD1 PHE A274 4.319 −24.701 45.509 1.00 68.18 A ATOM 1530 CD2 PHE A 274 3.502−26.955 45.413 1.00 67.97 A ATOM 1531 CE1 PHE A 274 3.155 −24.243 44.8961.00 68.69 A ATOM 1532 CE2 PHE A 274 2.332 −26.509 44.798 1.00 68.98 AATOM 1533 CZ PHE A 274 2.159 −25.149 44.539 1.00 69.46 A ATOM 1534 C PHEA 274 7.167 −28.430 47.229 1.00 60.36 A ATOM 1535 O PHE A 274 7.988−28.744 46.363 1.00 61.16 A ATOM 1536 N ALA A 275 7.456 −28.421 48.5261.00 58.26 A ATOM 1537 CA ALA A 275 8.781 −28.795 49.008 1.00 56.97 AATOM 1538 CB ALA A 275 8.768 −28.934 50.533 1.00 55.69 A ATOM 1539 C ALAA 275 9.220 −30.103 48.364 1.00 55.44 A ATOM 1540 O ALA A 275 8.450−31.060 48.301 1.00 56.27 A ATOM 1541 N ALA A 276 10.454 −30.136 47.8761.00 53.60 A ATOM 1542 CA ALA A 276 10.986 −31.336 47.243 1.00 51.53 AATOM 1543 CB ALA A 276 12.007 −30.960 46.179 1.00 52.85 A ATOM 1544 CALA A 276 11.633 −32.205 48.309 1.00 49.72 A ATOM 1545 O ALA A 27612.531 −32.997 48.023 1.00 50.08 A ATOM 1546 N PHE A 277 11.163 −32.04249.543 1.00 47.45 A ATOM 1547 CA PHE A 277 11.676 −32.785 50.687 1.0043.13 A ATOM 1548 CB PHE A 277 12.794 −31.986 51.358 1.00 42.71 A ATOM1549 CG PHE A 277 13.944 −31.678 50.448 1.00 41.75 A ATOM 1550 CD1 PHE A277 14.932 −32.626 50.213 1.00 40.75 A ATOM 1551 CD2 PHE A 277 14.021−30.449 49.798 1.00 39.70 A ATOM 1552 CE1 PHE A 277 15.981 −32.35449.343 1.00 41.23 A ATOM 1553 CE2 PHE A 277 15.063 −30.169 48.925 1.0039.82 A ATOM 1554 CZ PHE A 277 16.047 −31.122 48.696 1.00 40.51 A ATOM1555 C PHE A 277 10.550 −33.032 51.693 1.00 41.66 A ATOM 1556 O PHE A277 9.546 −32.322 51.709 1.00 40.72 A ATOM 1557 N PRO A 278 10.705−34.049 52.548 1.00 40.54 A ATOM 1558 CD PRO A 278 11.812 −35.020 52.6231.00 39.57 A ATOM 1559 CA PRO A 278 9.674 −34.352 53.544 1.00 39.44 AATOM 1560 CB PRO A 278 10.059 −35.748 54.005 1.00 38.76 A ATOM 1561 CGPRO A 278 11.561 −35.691 53.958 1.00 39.95 A ATOM 1562 C PRO A 278 9.670−33.336 54.693 1.00 39.01 A ATOM 1563 O PRO A 278 10.728 −32.960 55.2031.00 36.83 A ATOM 1564 N ILE A 279 8.481 −32.886 55.085 1.00 39.22 AATOM 1565 CA ILE A 279 8.351 −31.923 56.175 1.00 41.37 A ATOM 1566 CBILE A 279 7.673 −30.624 55.701 1.00 43.60 A ATOM 1567 CG2 ILE A 2797.599 −29.627 56.852 1.00 43.19 A ATOM 1568 CG1 ILE A 279 8.475 −30.02554.540 1.00 46.61 A ATOM 1569 CD1 ILE A 279 8.026 −28.640 54.110 1.0051.71 A ATOM 1570 C ILE A 279 7.549 −32.522 57.326 1.00 41.16 A ATOM1571 O ILE A 279 6.405 −32.952 57.150 1.00 41.86 A ATOM 1572 N TYR A 2808.160 −32.536 58.505 1.00 40.12 A ATOM 1573 CA TYR A 280 7.540 −33.10459.696 1.00 40.38 A ATOM 1574 CB TYR A 280 8.478 −34.138 60.315 1.0041.53 A ATOM 1575 CG TYR A 280 8.905 −35.230 59.371 1.00 43.98 A ATOM1576 CD1 TYR A 280 8.066 −36.309 59.097 1.00 45.26 A ATOM 1577 CE1 TYR A280 8.455 −37.316 58.216 1.00 45.37 A ATOM 1578 CD2 TYR A 280 10.147−35.181 58.738 1.00 44.40 A ATOM 1579 CE2 TYR A 280 10.544 −36.18057.856 1.00 45.61 A ATOM 1580 CZ TYR A 280 9.692 −37.244 57.600 1.0046.06 A ATOM 1581 OH TYR A 280 10.076 −38.231 56.724 1.00 47.42 A ATOM1582 C TYR A 280 7.192 −32.084 60.774 1.00 40.73 A ATOM 1583 O TYR A 2807.767 −30.995 60.837 1.00 36.63 A ATOM 1584 N VAL A 281 6.246 −32.47061.627 1.00 39.60 A ATOM 1585 CA VAL A 281 5.817 −31.655 62.750 1.0040.50 A ATOM 1586 CB VAL A 281 4.407 −31.066 62.534 1.00 40.77 A ATOM1587 CG1 VAL A 281 4.414 −30.113 61.360 1.00 40.09 A ATOM 1588 CG2 VAL A281 3.410 −32.181 62.302 1.00 44.48 A ATOM 1589 C VAL A 281 5.791−32.579 63.966 1.00 42.22 A ATOM 1590 O VAL A 281 5.274 −33.699 63.8981.00 41.86 A ATOM 1591 N TRP A 282 6.375 −32.121 65.066 1.00 42.62 AATOM 1592 CA TRP A 282 6.412 −32.907 66.290 1.00 45.45 A ATOM 1593 CBTRP A 282 7.432 −32.306 67.256 1.00 46.54 A ATOM 1594 CG TRP A 282 7.677−33.120 68.480 1.00 49.77 A ATOM 1595 CD2 TRP A 282 8.306 −34.405 68.5441.00 51.05 A ATOM 1596 CE2 TRP A 282 8.348 −34.781 69.905 1.00 51.72 AATOM 1597 CE3 TRP A 282 8.841 −35.276 67.585 1.00 51.26 A ATOM 1598 CD1TRP A 282 7.368 −32.780 69.763 1.00 50.66 A ATOM 1599 NE1 TRP A 2827.768 −33.771 70.626 1.00 50.98 A ATOM 1600 CZ2 TRP A 282 8.904 −35.99470.334 1.00 51.83 A ATOM 1601 CZ3 TRP A 282 9.395 −36.483 68.009 1.0051.83 A ATOM 1602 CH2 TRP A 282 9.421 −36.829 69.374 1.00 52.82 A ATOM1603 C TRP A 282 5.016 −32.860 66.890 1.00 46.75 A ATOM 1604 O TRP A 2824.639 −31.875 67.518 1.00 46.99 A ATOM 1605 N SER A 283 4.241 −33.92066.681 1.00 49.65 A ATOM 1606 CA SER A 283 2.876 −33.965 67.196 1.0053.00 A ATOM 1607 CB SER A 283 1.882 −34.042 66.036 1.00 54.08 A ATOM1608 OG SER A 283 2.123 −35.185 65.237 1.00 59.95 A ATOM 1609 C SER A283 2.612 −35.109 68.172 1.00 53.85 A ATOM 1610 O SER A 283 3.429−36.016 68.328 1.00 52.62 A ATOM 1611 N SER A 284 1.454 −35.048 68.8241.00 55.39 A ATOM 1612 CA SER A 284 1.047 −36.053 69.802 1.00 57.73 AATOM 1613 CB SER A 284 1.028 −35.436 71.197 1.00 57.15 A ATOM 1614 OGSER A 284 0.161 −34.317 71.228 1.00 57.89 A ATOM 1615 C SER A 284 −0.340−36.592 69.465 1.00 59.00 A ATOM 1616 O SER A 284 −1.101 −35.953 68.7391.00 58.48 A ATOM 1617 N LYS A 285 −0.671 −37.762 70.002 1.00 61.19 AATOM 1618 CA LYS A 285 −1.970 −38.368 69.732 1.00 62.47 A ATOM 1619 CBLYS A 285 −1.786 −39.800 69.222 1.00 62.13 A ATOM 1620 CG LYS A 285−3.076 −40.463 68.751 1.00 64.42 A ATOM 1621 CD LYS A 285 −2.824 −41.89468.297 1.00 65.81 A ATOM 1622 CE LYS A 285 −4.090 −42.549 67.762 1.0066.71 A ATOM 1623 NZ LYS A 285 −3.839 −43.956 67.323 1.00 68.96 A ATOM1624 C LYS A 285 −2.907 −38.372 70.938 1.00 62.88 A ATOM 1625 O LYS A285 −3.984 −37.776 70.889 1.00 63.71 A ATOM 1626 N THR A 286 −2.501−39.040 72.014 1.00 61.19 A ATOM 1627 CA THR A 286 −3.326 −39.127 73.2211.00 62.47 A ATOM 1628 CB THR A 286 −3.689 −37.724 73.773 1.00 62.13 AATOM 1629 OG1 THR A 286 −2.527 −37.110 74.345 1.00 19.86 A ATOM 1630 CG2THR A 286 −4.761 −37.835 74.848 1.00 19.86 A ATOM 1631 C THR A 286−4.628 −39.881 72.944 1.00 62.88 A ATOM 1632 O THR A 286 −4.619 −41.00872.445 1.00 63.71 A ATOM 1633 N LYS A 328 −2.207 −41.457 78.617 1.0067.81 A ATOM 1634 CA LYS A 328 −1.228 −41.967 77.661 1.00 68.19 A ATOM1635 CB LYS A 328 −1.633 −43.360 77.175 1.00 70.00 A ATOM 1636 CG LYS A328 −1.698 −44.415 78.266 1.00 73.35 A ATOM 1637 CD LYS A 328 −2.084−45.771 77.687 1.00 75.20 A ATOM 1638 CE LYS A 328 −2.144 −46.846 78.7621.00 76.19 A ATOM 1639 NZ LYS A 328 −2.470 −48.180 78.184 1.00 77.71 AATOM 1640 C LYS A 328 −1.094 −41.040 76.458 1.00 67.51 A ATOM 1641 O LYSA 328 −2.084 −40.710 75.802 1.00 67.72 A ATOM 1642 N THR A 329 0.136−40.626 76.168 1.00 65.72 A ATOM 1643 CA THR A 329 0.391 −39.740 75.0391.00 64.09 A ATOM 1644 CB THR A 329 0.655 −38.300 75.504 1.00 63.35 AATOM 1645 OG1 THR A 329 −0.463 −37.831 76.268 1.00 62.53 A ATOM 1646 CG2THR A 329 0.865 −37.390 74.302 1.00 62.46 A ATOM 1647 C THR A 329 1.600−40.207 74.241 1.00 63.16 A ATOM 1648 O THR A 329 2.645 −40.524 74.8111.00 62.83 A ATOM 1649 N VAL A 330 1.455 −40.250 72.920 1.00 62.00 AATOM 1650 CA VAL A 330 2.553 −40.674 72.061 1.00 60.58 A ATOM 1651 CBVAL A 330 2.150 −41.858 71.159 1.00 60.87 A ATOM 1652 CG1 VAL A 3303.398 −42.483 70.549 1.00 60.39 A ATOM 1653 CG2 VAL A 330 1.366 −42.88971.957 1.00 60.97 A ATOM 1654 C VAL A 330 3.007 −39.522 71.173 1.0059.36 A ATOM 1655 O VAL A 330 2.220 −38.958 70.413 1.00 58.20 A ATOM1656 N TRP A 331 4.286 −39.180 71.279 1.00 59.04 A ATOM 1657 CA TRP A331 4.865 −38.095 70.497 1.00 58.50 A ATOM 1658 CB TRP A 331 5.615−37.132 71.420 1.00 60.31 A ATOM 1659 CG TRP A 331 4.918 −35.828 71.6491.00 62.59 A ATOM 1660 CD2 TRP A 331 4.313 −35.382 72.867 1.00 63.34 AATOM 1661 CE2 TRP A 331 3.804 −34.085 72.629 1.00 64.50 A ATOM 1662 CE3TRP A 331 4.152 −35.951 74.136 1.00 63.96 A ATOM 1663 CD1 TRP A 3314.753 −34.815 70.746 1.00 64.47 A ATOM 1664 NE1 TRP A 331 4.086 −33.76471.327 1.00 64.58 A ATOM 1665 CZ2 TRP A 331 3.145 −33.346 73.616 1.0064.39 A ATOM 1666 CZ3 TRP A 331 3.497 −35.216 75.118 1.00 66.44 A ATOM1667 CH2 TRP A 331 3.001 −33.925 74.851 1.00 66.38 A ATOM 1668 C TRP A331 5.821 −38.624 69.432 1.00 57.08 A ATOM 1669 O TRP A 331 6.648−39.496 69.701 1.00 55.66 A ATOM 1670 N ASP A 332 5.701 −38.090 68.2211.00 55.73 A ATOM 1671 CA ASP A 332 6.569 −38.495 67.125 1.00 54.33 AATOM 1672 CB ASP A 332 6.197 −39.898 66.644 1.00 55.67 A ATOM 1673 CGASP A 332 7.412 −40.726 66.271 1.00 57.50 A ATOM 1674 OD1 ASP A 3328.260 −40.236 65.496 1.00 57.91 A ATOM 1675 OD2 ASP A 332 7.517 −41.87366.754 1.00 59.97 A ATOM 1676 C ASP A 332 6.454 −37.510 65.962 1.0052.80 A ATOM 1677 O ASP A 332 5.546 −36.674 65.927 1.00 51.02 A ATOM1678 N TRP A 333 7.383 −37.609 65.017 1.00 50.33 A ATOM 1679 CA TRP A333 7.366 −36.739 63.852 1.00 48.17 A ATOM 1680 CB TRP A 333 8.682−36.842 63.083 1.00 45.68 A ATOM 1681 CG TRP A 333 9.867 −36.381 63.8521.00 43.61 A ATOM 1682 CD2 TRP A 333 10.177 −35.031 64.222 1.00 41.57 AATOM 1683 CE2 TRP A 333 11.398 −35.063 64.931 1.00 42.05 A ATOM 1684 CE3TRP A 333 9.542 −33.798 64.023 1.00 40.61 A ATOM 1685 CD1 TRP A 33310.881 −37.155 64.339 1.00 42.81 A ATOM 1686 NE1 TRP A 333 11.805−36.372 64.987 1.00 43.41 A ATOM 1687 CZ2 TRP A 333 12.001 −33.90565.445 1.00 40.00 A ATOM 1688 CZ3 TRP A 333 10.144 −32.642 64.536 1.0039.42 A ATOM 1689 CH2 TRP A 333 11.360 −32.709 65.237 1.00 37.87 A ATOM1690 C TRP A 333 6.226 −37.168 62.945 1.00 47.93 A ATOM 1691 O TRP A 3336.101 −38.344 62.610 1.00 48.69 A ATOM 1692 N GLU A 334 5.386 −36.21762.560 1.00 47.26 A ATOM 1693 CA GLU A 334 4.273 −36.520 61.674 1.0047.77 A ATOM 1694 CB GLU A 334 2.963 −35.974 62.241 1.00 47.55 A ATOM1695 CG GLU A 334 1.729 −36.494 61.521 1.00 48.73 A ATOM 1696 CD GLU A334 0.459 −35.807 61.974 1.00 52.26 A ATOM 1697 OE1 GLU A 334 0.229−35.728 63.198 1.00 51.80 A ATOM 1698 OE2 GLU A 334 −0.313 −35.34861.105 1.00 54.38 A ATOM 1699 C GLU A 334 4.545 −35.889 60.313 1.0047.58 A ATOM 1700 O GLU A 334 4.742 −34.678 60.206 1.00 46.83 A ATOM1701 N LEU A 335 4.565 −36.719 59.277 1.00 47.77 A ATOM 1702 CA LEU A335 4.817 −36.248 57.922 1.00 48.66 A ATOM 1703 CB LEU A 335 5.043−37.443 56.991 1.00 47.95 A ATOM 1704 CG LEU A 335 5.398 −37.137 55.5351.00 47.37 A ATOM 1705 CD1 LEU A 335 6.661 −36.288 55.479 1.00 45.63 AATOM 1706 CD2 LEU A 335 5.593 −38.447 54.776 1.00 47.26 A ATOM 1707 CLEU A 335 3.657 −35.403 57.405 1.00 48.95 A ATOM 1708 O LEU A 335 2.520−35.863 57.344 1.00 50.26 A ATOM 1709 N MET A 336 3.952 −34.159 57.0461.00 49.79 A ATOM 1710 CA MET A 336 2.942 −33.249 56.523 1.00 50.82 AATOM 1711 CB MET A 336 3.183 −31.829 57.045 1.00 51.61 A ATOM 1712 CGMET A 336 3.193 −31.704 58.565 1.00 51.96 A ATOM 1713 SD MET A 336 1.620−32.164 59.335 1.00 54.16 A ATOM 1714 CE MET A 336 1.964 −33.821 59.7591.00 52.24 A ATOM 1715 C MET A 336 3.064 −33.270 55.004 1.00 51.69 AATOM 1716 O MET A 336 2.179 −32.808 54.288 1.00 50.87 A ATOM 1717 N ASNA 337 4.181 −33.822 54.539 1.00 53.30 A ATOM 1718 CA ASN A 337 4.500−33.952 53.119 1.00 55.08 A ATOM 1719 CB ASN A 337 3.257 −34.343 52.3181.00 55.89 A ATOM 1720 CG ASN A 337 3.582 −34.679 50.885 1.00 57.84 AATOM 1721 OD1 ASN A 337 4.363 −35.590 50.617 1.00 59.85 A ATOM 1722 ND2ASN A 337 2.988 −33.942 49.950 1.00 60.12 A ATOM 1723 C ASN A 337 5.107−32.686 52.529 1.00 52.82 A ATOM 1724 O ASN A 337 6.305 −32.640 52.2581.00 40.66 A ATOM 1725 C5* NEC N 1 18.101 −14.744 53.515 1.00 32.09 NATOM 1726 O5* NEC N 1 16.931 −15.464 53.805 1.00 33.94 N ATOM 1727 N5*NEC N 1 18.132 −13.321 53.673 1.00 34.59 N ATOM 1728 C51 NEC N 1 18.051−12.476 52.504 1.00 39.19 N ATOM 1729 C52 NEC N 1 16.800 −11.615 52.6011.00 38.66 N ATOM 1730 C4* NEC N 1 19.356 −15.441 53.081 1.00 32.40 NATOM 1731 O4* NEC N 1 19.996 −16.008 54.191 1.00 31.40 N ATOM 1732 C3*NEC N 1 19.175 −16.570 52.078 1.00 30.36 N ATOM 1733 O3* NEC N 1 19.248−16.137 50.739 1.00 31.36 N ATOM 1734 C2* NEC N 1 20.257 −17.579 52.5031.00 33.06 N ATOM 1735 O2* NEC N 1 21.430 −17.328 51.832 1.00 30.18 NATOM 1736 C1* NEC N 1 20.339 −17.393 54.061 1.00 29.68 N ATOM 1737 N9NEC N 1 19.362 −18.313 54.711 1.00 31.55 N ATOM 1738 C8 NEC N 1 18.167−18.005 55.335 1.00 29.83 N ATOM 1739 N7 NEC N 1 17.556 −19.059 55.8071.00 30.83 N ATOM 1740 C5 NEC N 1 18.399 −20.121 55.490 1.00 30.97 NATOM 1741 C6 NEC N 1 18.336 −21.533 55.729 1.00 31.98 N ATOM 1742 N6 NECN 1 17.315 −22.108 56.374 1.00 29.34 N ATOM 1743 N1 NEC N 1 19.367−22.326 55.284 1.00 32.04 N ATOM 1744 C2 NEC N 1 20.402 −21.758 54.6331.00 31.64 N ATOM 1745 N3 NEC N 1 20.574 −20.454 54.344 1.00 31.62 NATOM 1746 C4 NEC N 1 19.524 −19.676 54.805 1.00 30.07 N ATOM 1747 OH2WAT W 1 20.447 −24.737 56.287 1.00 24.64 W ATOM 1748 OH2 WAT W 2 14.568−14.841 53.450 1.00 22.42 W ATOM 1749 OH2 WAT W 3 19.296 −8.909 53.2161.00 34.41 W ATOM 1750 OH2 WAT W 4 −9.791 −31.932 65.071 1.00 60.00 WATOM 1751 OH2 WAT W 5 12.411 −2.377 65.438 1.00 32.85 W ATOM 1752 OH2WAT W 6 12.886 −28.142 64.918 1.00 28.26 W ATOM 1753 OH2 WAT W 8 20.659−2.334 63.849 1.00 27.95 W ATOM 1754 OH2 WAT W 9 14.414 −21.453 57.2221.00 33.19 W ATOM 1755 OH2 WAT W 10 16.630 −18.755 50.263 1.00 39.77 WATOM 1756 OH2 WAT W 11 28.580 −23.175 60.300 1.00 31.58 W ATOM 1757 OH2WAT W 12 25.144 −21.263 53.006 1.00 35.45 W ATOM 1758 OH2 WAT W 1314.906 −23.981 58.677 1.00 26.25 W ATOM 1759 OH2 WAT W 14 27.806 −3.02862.773 1.00 37.16 W ATOM 1760 OH2 WAT W 15 27.329 −27.459 54.451 1.0024.48 W ATOM 1761 OH2 WAT W 17 13.107 −18.978 49.611 1.00 38.19 W ATOM1762 OH2 WAT W 18 17.985 −2.488 63.260 1.00 28.53 W ATOM 1763 OH2 WAT W19 9.101 −7.920 66.355 1.00 30.77 W ATOM 1764 OH2 WAT W 20 27.388−32.822 56.057 1.00 39.96 W ATOM 1765 OH2 WAT W 21 −0.940 −7.731 54.6531.00 44.25 W ATOM 1766 OH2 WAT W 22 32.699 −22.173 48.909 1.00 59.25 WATOM 1767 OH2 WAT W 23 15.056 −18.808 56.978 1.00 22.64 W ATOM 1768 OH2WAT W 24 17.946 −21.501 66.688 1.00 26.54 W ATOM 1769 OH2 WAT W 2513.721 −2.135 62.627 1.00 39.41 W ATOM 1770 OH2 WAT W 26 12.394 −25.57172.991 1.00 35.33 W ATOM 1771 OH2 WAT W 27 20.012 −29.619 61.103 1.0025.85 W ATOM 1772 OH2 WAT W 28 17.936 −20.651 52.066 1.00 28.66 W ATOM1773 OH2 WAT W 29 0.586 −12.872 44.960 1.00 34.56 W ATOM 1774 OH2 WAT W30 15.573 −27.732 68.375 1.00 44.80 W ATOM 1775 OH2 WAT W 31 22.608−19.886 52.600 1.00 34.40 W ATOM 1776 OH2 WAT W 32 1.893 −11.084 65.7971.00 34.78 W ATOM 1777 OH2 WAT W 33 24.264 0.954 56.229 1.00 35.33 WATOM 1778 OH2 WAT W 34 16.289 −35.704 59.755 1.00 42.89 W ATOM 1779 OH2WAT W 36 11.669 −3.349 62.349 1.00 47.32 W ATOM 1780 OH2 WAT W 37 22.055−1.433 66.332 1.00 37.36 W ATOM 1781 OH2 WAT W 38 28.886 −27.612 51.7811.00 35.01 W ATOM 1782 OH2 WAT W 39 12.130 −16.233 51.524 1.00 28.89 WATOM 1783 OH2 WAT W 40 28.128 −29.763 58.789 1.00 38.41 W ATOM 1784 OH2WAT W 41 10.890 −4.366 67.600 1.00 39.52 W ATOM 1785 OH2 WAT W 42 2.443−14.674 47.358 1.00 30.52 W ATOM 1786 OH2 WAT W 43 21.091 −12.400 51.6271.00 38.41 W ATOM 1787 OH2 WAT W 44 31.286 −29.871 68.533 1.00 48.27 WATOM 1788 OH2 WAT W 45 11.801 −3.011 69.667 1.00 31.67 W ATOM 1789 OH2WAT W 46 18.867 −8.703 69.847 1.00 51.34 W ATOM 1790 OH2 WAT W 47 28.938−32.732 61.259 1.00 42.94 W ATOM 1791 OH2 WAT W 48 7.647 −12.538 73.4931.00 43.37 W ATOM 1792 OH2 WAT W 49 15.138 −31.436 60.017 1.00 34.37 WATOM 1793 OH2 WAT W 51 14.648 −30.717 65.458 1.00 59.12 W ATOM 1794 OH2WAT W 52 −0.388 −35.583 57.653 1.00 59.38 W ATOM 1795 OH2 WAT W 54 4.4080.061 38.685 1.00 69.58 W ATOM 1796 OH2 WAT W 55 4.392 −39.653 59.5771.00 49.80 W ATOM 1797 OH2 WAT W 56 10.651 −17.908 48.691 1.00 48.73 WATOM 1798 OH2 WAT W 57 8.040 −18.417 49.106 1.00 52.79 W ATOM 1799 OH2WAT W 59 15.761 −30.831 45.009 1.00 79.83 W ATOM 1800 OH2 WAT W 60 2.973−8.078 63.778 1.00 41.27 W ATOM 1801 OH2 WAT W 61 25.327 −5.578 59.2641.00 45.94 W ATOM 1802 OH2 WAT W 63 6.882 −22.869 51.273 1.00 42.05 WATOM 1803 OH2 WAT W 64 35.748 −20.905 61.008 1.00 69.02 W ATOM 1804 OH2WAT W 65 −4.257 −31.908 65.453 1.00 57.93 W ATOM 1805 OH2 WAT W 6710.789 −6.923 67.951 1.00 40.87 W ATOM 1806 OH2 WAT W 68 30.679 −29.80951.561 1.00 47.80 W ATOM 1807 OH2 WAT W 70 14.686 −36.386 52.025 1.0058.07 W ATOM 1808 OH2 WAT W 71 23.286 −34.743 65.028 1.00 64.95 W ATOM1809 OH2 WAT W 74 13.476 −24.792 47.142 1.00 44.00 W ATOM 1810 OH2 WAT W75 27.424 −8.021 52.318 1.00 50.04 W ATOM 1811 OH2 WAT W 76 17.185−11.984 47.384 1.00 68.65 W ATOM 1812 OH2 WAT W 77 5.622 −29.583 68.2261.00 54.52 W ATOM 1813 OH2 WAT W 80 18.815 −24.842 68.793 1.00 40.43 WATOM 1814 OH2 WAT W 81 21.343 5.380 55.616 1.00 33.74 W ATOM 1815 OH2WAT W 82 16.590 −29.691 67.097 1.00 46.02 W ATOM 1816 OH2 WAT W 8320.194 −21.601 51.021 1.00 36.86 W ATOM 1817 OH2 WAT W 85 14.715 −27.83670.942 1.00 51.90 W ATOM 1818 OH2 WAT W 86 13.436 −29.582 72.049 1.0050.86 W ATOM 1819 OH2 WAT W 87 20.259 −10.157 51.078 1.00 44.59 W ATOM1820 OH2 WAT W 88 26.900 −0.804 66.233 1.00 39.07 W ATOM 1821 OH2 WAT W89 4.820 −9.903 73.149 1.00 40.81 W ATOM 1822 OH2 WAT W 90 −0.281−12.552 65.555 1.00 49.05 W ATOM 1823 OH2 WAT W 92 12.724 −5.568 71.5861.00 53.51 W ATOM 1824 OH2 WAT W 93 7.826 −43.002 69.415 1.00 54.98 WATOM 1825 OH2 WAT W 95 20.986 −12.197 43.875 1.00 63.86 W ATOM 1826 OH2WAT W 96 32.091 −11.891 74.247 1.00 56.91 W ATOM 1827 OH2 WAT W 9714.437 −24.757 74.268 1.00 57.27 W ATOM 1828 OH2 WAT W 98 30.503 −21.73347.723 1.00 55.05 W ATOM 1829 OH2 WAT W 100 −2.534 −12.377 60.294 1.0046.67 W ATOM 1830 OH2 WAT W 101 6.007 −40.679 73.453 1.00 60.10 W ATOM1831 OH2 WAT W 103 9.741 −6.705 70.312 1.00 51.80 W ATOM 1832 OH2 WAT W104 18.884 −23.389 46.288 1.00 42.13 W ATOM 1833 OH2 WAT W 105 8.278−40.462 72.427 1.00 54.94 W ATOM 1834 OH2 WAT W 106 30.185 −18.89072.770 1.00 50.94 W ATOM 1835 OH2 WAT W 110 31.599 −30.829 60.833 1.0045.28 W ATOM 1836 OH2 WAT W 111 7.128 −4.100 40.767 1.00 44.90 W ATOM1837 OH2 WAT W 112 5.094 −27.238 75.588 1.00 59.12 W ATOM 1838 OH2 WAT W113 18.993 −31.333 62.938 1.00 38.54 W ATOM 1839 OH2 WAT W 117 27.880−23.843 50.359 1.00 50.76 W ATOM 1840 OH2 WAT W 118 −1.684 −13.82263.153 1.00 59.71 W ATOM 1841 OH2 WAT W 123 17.812 −27.177 71.950 1.0060.66 W ATOM 1842 OH2 WAT W 125 2.093 −21.356 71.451 1.00 48.69 W ATOM1843 OH2 WAT W 126 −3.442 −17.247 61.865 1.00 54.19 W ATOM 1844 OH2 WATW 127 1.759 −10.946 45.001 1.00 41.89 W ATOM 1845 OH2 WAT W 128 −8.441−42.749 77.832 1.00 70.82 W ATOM 1846 OH2 WAT W 129 7.800 −36.923 51.2171.00 53.53 W ATOM 1847 OH2 WAT W 130 10.080 −40.723 68.230 1.00 53.30 WATOM 1848 OH2 WAT W 131 26.285 1.358 67.018 1.00 48.30 W ATOM 1849 OH2WAT W 132 22.963 −11.064 60.039 1.00 90.89 W ATOM 1850 OH2 WAT W 13410.353 −4.541 64.097 1.00 56.18 W ATOM 1851 OH2 WAT W 137 3.597 −17.83845.819 1.00 54.21 W ATOM 1852 OH2 WAT W 139 −3.897 −18.680 64.339 1.0054.18 W ATOM 1853 OH2 WAT W 140 16.956 −20.939 44.880 1.00 69.00 W ATOM1854 OH2 WAT W 142 14.738 −38.326 59.530 1.00 58.88 W ATOM 1855 OH2 WATW 145 14.855 −23.001 76.133 1.00 43.96 W ATOM 1856 OH2 WAT W 147 20.215−26.877 69.337 1.00 64.57 W ATOM 1857 OH2 WAT W 155 16.455 −32.06462.139 1.00 47.73 W ATOM 1858 OH2 WAT W 156 18.383 −23.330 74.540 1.0059.11 W ATOM 1859 OH2 WAT W 160 26.823 −3.191 54.242 1.00 60.75 W ATOM1860 OH2 WAT W 161 34.156 −28.509 60.729 1.00 70.43 W ATOM 1861 OH2 WATW 170 21.242 −28.298 75.598 1.00 51.15 W ATOM 1862 OH2 WAT W 171 23.3624.612 56.588 1.00 46.95 W ATOM 1863 OH2 WAT W 172 −6.396 −44.988 78.1591.00 54.39 W ATOM 1864 OH2 WAT W 173 18.594 −21.905 72.366 1.00 56.44 WATOM 1865 OH2 WAT W 174 10.847 −2.441 73.845 1.00 45.58 W ATOM 1866 OH2WAT W 180 12.073 −24.440 77.663 1.00 65.15 W ATOM 1867 OH2 WAT W 18611.906 −40.592 66.069 1.00 71.20 W ATOM 1868 OH2 WAT W 194 25.784−30.114 73.492 1.00 56.89 W ATOM 1869 OH2 WAT W 195 3.140 −16.054 72.5711.00 59.02 W ATOM 1870 OH2 WAT W 196 4.981 −17.689 72.622 1.00 95.43 WATOM 1871 OH2 WAT W 197 18.315 −22.761 69.952 1.00 56.52 W ATOM 1872 OH2WAT W 198 −0.003 −0.358 39.802 1.00 51.25 W ATOM 1873 OH2 WAT W 1998.744 −26.111 49.896 1.00 66.37 W ATOM 1874 OH2 WAT W 202 24.098 2.93158.413 1.00 46.53 W ATOM 1875 OH2 WAT W 203 16.861 −34.165 43.335 1.0081.19 W ATOM 1876 OH2 WAT W 204 19.382 −26.393 74.298 1.00 67.51 W ATOM1877 OH2 WAT W 206 24.225 3.592 53.248 1.00 58.61 W ATOM 1878 OH2 WAT W207 31.912 −18.264 54.777 1.00 56.00 W ATOM 1879 OH2 WAT W 208 11.384−32.171 71.466 1.00 80.10 W ATOM 1880 OH2 WAT W 211 24.900 −11.18343.677 1.00 52.88 W ATOM 1881 OH2 WAT W 212 16.768 −16.252 49.717 1.0053.52 W ATOM 1882 OH2 WAT W 214 10.711 −26.840 77.276 1.00 64.18 W ATOM1883 OH2 WAT W 215 28.159 −2.789 65.544 1.00 53.99 W ATOM 1884 OH2 WAT W216 −5.027 −22.160 56.481 1.00 67.91 W ATOM 1885 OH2 WAT W 217 5.564−10.768 41.516 1.00 62.02 W ATOM 1886 OH2 WAT W 218 34.758 −32.99144.233 1.00 67.86 W ATOM 1887 OH2 WAT W 219 15.383 −35.199 64.512 1.0057.58 W ATOM 1888 OH2 WAT W 220 11.042 −0.993 71.541 1.00 40.57 W ATOM1889 OH2 WAT W 221 −1.373 −35.364 55.318 1.00 63.40 W ATOM 1890 OH2 WATW 222 −1.414 −28.056 71.313 1.00 51.06 W ATOM 1891 OH2 WAT W 223 0.579−15.614 44.883 1.00 57.51 W ATOM 1892 OH2 WAT W 224 20.213 −21.40945.285 1.00 59.50 W ATOM 1893 OH2 WAT W 226 16.554 −23.850 44.983 1.0053.48 W ATOM 1894 OH2 WAT W 227 31.301 −14.218 66.543 1.00 67.17 W ATOM1895 OH2 WAT W 229 17.319 −12.439 44.761 1.00 62.52 W ATOM 1896 OH2 WATW 230 33.266 −19.036 74.435 1.00 52.88 W ATOM 1897 OH2 WAT W 231 −4.526−13.517 58.476 1.00 63.03 W ATOM 1898 OH2 WAT W 232 25.471 −17.82651.113 1.00 50.66 W ATOM 1899 OH2 WAT W 233 30.569 −27.352 65.414 1.0058.69 W ATOM 1900 OH2 WAT W 234 32.392 −27.907 51.765 1.00 56.34 W ATOM1901 OH2 WAT W 235 18.193 −0.620 46.316 1.00 66.93 W ATOM 1902 OH2 WAT W236 28.994 −9.891 51.100 1.00 57.32 W ATOM 1903 OH2 WAT W 237 14.361−17.969 45.938 1.00 67.16 W ATOM 1904 OH2 WAT W 239 29.694 −37.64343.980 1.00 59.17 W ATOM 1905 OH2 WAT W 240 9.220 −28.951 72.297 1.0053.08 W ATOM 1906 OH2 WAT W 242 7.337 −31.202 73.280 1.00 80.10 W ATOM1907 OH2 WAT W 243 11.834 −33.939 69.589 1.00 80.10 W ATOM 1908 OH2 WATW 244 11.476 −0.583 66.761 1.00 80.10 W

It will be understood that various details of the invention can bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation—the invention being defined by theclaims.

1. A crystallized complex of a GRP94 ligand binding domain polypeptideand a ligand, wherein: (a) the GRP94 ligand binding domain polypeptideconsists of amino acids 48-316 of SEQ ID NO: 6; (b) the ligand comprisesN-ethylcarboxamidoadenosine (NECA); and (c) the crystallized complex hasa crystalline form selected from the group consisting of: (i) acrystalline form with unit cell lattice constants of a=99.889 Å,b=89.614 Å, c=60.066 Å; β=90.132°; space group symmetry C2; and 2GRP94+NECA complexes in the asymmetric unit; and (ii) a crystalline formwith lattice constants of a=89.200 Å, b=99.180 Å, c=63.071 Å;α=β=γ=90.0°; space group symmetry C222₍₁₎; and 1 GRP94+NECA complex inthe asymmetric unit.
 2. A method of generating a crystallized GRP94ligand binding domain polypeptide complexed toN-ethylcarboxamidoadenosine (NECA), the method comprising: (a) preparinga solution containing 30 mg/ml of a GRP94 ligand binding domainpolypeptide consisting of amino acids 48-316 of SEQ ID NO: 6 in 10 mMTris-HCl, pH 7.6, 1 mM DTT, 100 mM NaCl and three molar equivalents ofNECA to the GRP94 ligand binding domain polypeptide; and (b) growing acrystal at 18° C. using the hanging drop method by mixing equal volumesof the solution of (a) with a reservoir solution consisting of 100 mMTris-HCl, pH 7.6, 150-250 mM MgCl₂, and 33-38% polyethylene glycol 400(PEG 400), whereby a crystallized GRP94 ligand binding domainpolypeptide complexed to NECA is generated.
 3. A crystalline compositionproduced by the method of claim 2.